Compositions and methods for preventing or treating muscle conditions

ABSTRACT

Provided herein are compositions for preventing or treating muscle conditions such as muscle damage, injury, or atrophy. In some embodiments, the compositions comprise a prostaglandin E2 (PGE2) compound and a myotoxin. In some embodiments, the muscle damage, injury, or atrophy is the result of a nerve injury, a surgical procedure, or a traumatic injury. Methods of promoting muscle regeneration and methods of increasing muscle mass are also provided herein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of PCT Application No.PCT/US2018/036727, filed Jun. 8, 2018, which claims priority to U.S.Provisional Application No. 62/517,758, filed Jun. 9, 2017, thedisclosure of which is hereby incorporated by reference in its entiretyfor all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No.AG020961, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Muscle injuries are an extremely common phenomenon which range frombeing relatively mild to extremely severe and can take many forms. Inaddition, muscle injuries arise from any number of causes. For example,muscles are often incidentally injured during surgical procedures (e.g.,surgical treatments). This is especially apparent in the context ofsurgical procedures such as Caesarean sections and joint replacementsurgeries (e.g., knee and hip replacement surgeries). Furthermore, manymuscle injuries are the result of trauma and accidental events thatproduce cutting, compression, and/or crushing of muscle tissue. Inaddition, many muscle injuries are the consequence of immobilization(e.g., limb immobilization) and/or nerve injuries (e.g., peripheralnerve injuries).

Peripheral nerve injuries (PNI) are a common result of trauma orimmobilization due to illness and can produce severe motor deficits thatultimately impact the physical, psychological, and social well-being ofthose affected. In particular, peripheral nerves are prevalently injuredin combat from high velocity gunshot wounds and explosive fragments.Furthermore, combat PNIs are increasingly common because improvements inbody armor and rapid evacuations allow more soldiers to survive severeextremity trauma. PNIs occurred in 8% of UK combat casualties from theIraq and Afghanistan conflicts. Of those with combat PNIs, only about 9%return to full duty.

Compression PNIs (such as carpal tunnel syndrome (CTS)) are a categoryof nerve injury caused by constriction of the nerve. Compression PNIsare especially common in the military veteran population. For example,in 2007-2008 120,000 veterans received a diagnosis of CTS and 10,000 ofthem underwent carpal tunnel release.

A primary morbidity after PNI (either due to combat or compression) ismuscle atrophy that occurs when a muscle is denervated. Recovery ofdenervated muscle is a complex process that is not fully understood;however intrinsic regenerative factors of the muscle are known to becritical and can be influenced by factors such as age. For those withsevere CTS, the denervated muscle is the abductor pollicis brevis (APB),which brings the thumb out of the plane of the palm and is integral tomany fine motor activities (FIG. 1). To date, the only means of medicalintervention is by surgery to release the band constricting the mediannerve. This allows for regeneration of the motor nerve and potentialrecovery of the muscle. Unfortunately, many of those with severe CTShave poor functional recovery even after the nerve has been released.

There is a need for new therapies that improve the recovery of musclefunction following muscle and nerve injuries. The present inventionsatisfies this need, and provides related advantages as well.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, provided herein is a composition for preventing ortreating a muscle condition. In some embodiments, the compositioncomprises a prostaglandin E2 (PGE2) compound and a myotoxin.

In a second aspect, provided herein is a pharmaceutical composition. Insome embodiments, the pharmaceutical composition comprises a compositiondescribed herein that comprises a PGE2 compound and a myotoxin.

In a third aspect, provided herein is a method for promoting muscleregeneration and/or increasing muscle mass in a subject in need thereof.In some embodiments, the method comprises administering a combination ofa PGE2 compound and a myotoxin to the subject.

In a fourth aspect, provided herein is a method for preventing ortreating a muscle condition in a subject in need thereof. In someembodiments, the method comprises administering a combination of a PGE2compound and a myotoxin to the subject. In other embodiments, the methodcomprises administering a PGE2 receptor agonist and a myotoxin to thesubject.

In fifth aspect, provided herein is a kit for promoting muscleregeneration, increasing muscle strength, and/or increasing muscle massin a subject in need thereof, or for preventing or treating a musclecondition in a subject in need thereof. In some embodiments, the kitcomprises a composition described herein that comprises a combination ofa PGE2 compound and a myotoxin. In other embodiments, the kit comprisesa pharmaceutical composition described herein.

Described herein is a composition for preventing or treating a musclecondition, the composition comprising a prostaglandin E2 (PGE2) compoundand a myotoxin. In some embodiments, the PGE2 compound is selected fromthe group consisting of PGE2, a PGE2 prodrug, a PGE2 receptor agonist, acompound that attenuates PGE2 catabolism, a compound that neutralizesPGE2 inhibition, a derivative thereof, an analog thereof, and acombination thereof. In some embodiments, the PGE2 receptor agonistcomprises a compound of Formula (I), a derivative thereof, apharmaceutically acceptable salt thereof, a solvate thereof, astereoisomer thereof, or a combination thereof,

-   -   wherein ring A is a substituted 4- to 6-membered cycloalkyl ring        or a substituted 4- to 6-membered cycloalkenyl ring that        comprises substituents R¹ and R² that are independently selected        from the group consisting of substituted C₁-C₁₀ alkyl and        substituted C₂-C₁₀ alkenyl, and ring A further comprises one or        more additional substituents.

In some cases, ring A is a substituted cyclopentyl ring or a substitutedcyclopentenyl ring. In some cases, the one or more additionalsubstituents on ring A are selected from the group consisting ofdeuterium, hydroxy, amino, oxo, Cl-C6 alkyl, and halogen. In some cases,the one or more additional substituents on ring A are hydroxy or oxo. Insome embodiments, ring A has two additional substituents that are takentogether to form a covalent bond to form a heterocycloalkyl ring. Insome embodiments, ring A is selected from the group consisting of

In some embodiments, ring A is selected from the group consisting of

In some embodiments, ring A is

In some embodiments, R¹ is substituted C₁-C₁₀ alkyl. In someembodiments, R¹ is substituted C₂-C₁₀ alkenyl. In some embodiments, thesubstituent on R¹ is selected from the group consisting of deuterium,hydroxy, oxo, C₁-C₆ alkyl, —COOR³, and halogen, wherein R³ is hydrogenor C₁-C₆ alkyl. In some embodiments, R¹ is selected from the groupconsisting of

In some embodiments, R¹ is selected from the group consisting of

In some embodiments, R¹ is

In some embodiments, R² is substituted C₁-C₁₀ alkyl.

In some embodiments, R² is substituted C₂-C₁₀ alkenyl. In someembodiments, the substituent on R² is selected from the group consistingof deuterium, hydroxy, oxo, C₁-C₆ alkyl, —COOR³, and halogen, wherein R³is hydrogen or C₁-C₆ alkyl. In some embodiments, R² is selected from thegroup consisting of

In some embodiments, R² is selected from the group consisting of

In some embodiments, R² is

In some embodiments, the compound of Formula (I), the pharmaceuticallyacceptable salt thereof, the solvate thereof, or the stereoisomerthereof is a compound of Formula (Ia), Formula (Ib), Formula (Ic), orFormula (Id), or a pharmaceutically acceptable salt, solvate, orstereoisomer thereof:

In some embodiments, the compound of Formula (I), the pharmaceuticallyacceptable salt thereof, the solvate thereof, or the stereoisomerthereof is a compound of Formula (Id), or a pharmaceutically acceptablesalt, solvate, or stereoisomer thereof.

In some embodiments, the PGE2 derivative comprises 16,16-dimethylprostaglandin E2. In some embodiments, the compound that attenuates PGE2catabolism comprises a compound, neutralizing peptide, or neutralizingantibody that inactivates or blocks 15-hydroxyprostaglandindehydrogenase (15-PGDH) or inactivates or blocks a prostaglandintransporter (PGT or SLCO2A1). In some embodiments, the PGE2 compound isPGE2.

In some embodiments, the myotoxin is selected from the group consistingof an anesthetic, a divalent cation, snake venom, lizard venom, beevenom, and a combination thereof. In some embodiments, the anesthetic isselected from the group consisting of an amino-amide anesthetic, anamino-ester anesthetic, and a combination thereof. In some embodiments,the amino-amide anesthetic is selected from the group consisting ofbupivacaine, levobupivacaine, articaine, ropivacaine, butanilicaine,carticaine, dibucaine, etidocaine, lidocaine, mepivacaine, prilocaine,trimecaine, and a combination thereof. In some embodiments, theamino-ester anesthetic is selected from the group consisting of anaminobenzoic acid ester anesthetic, a benzoic acid ester anesthetic, anda combination thereof. In some embodiments, the aminobenzoic acid esteranesthetic is selected from the group consisting of benzocaine,butacaine, butamben, chloroprocaine, dimethocaine, lucaine, meprylcaine,metabutethamine, metabutoxycaine, nitracaine, orthocaine, propoxycaine,procaine, proxymetacaine, procaine, tetracaine, and a combinationthereof. In some embodiments, the benzoic acid ester anesthetic isselected from the group consisting of amylocaine, cocaine,cyclomethycaine, α-eucaine, β-eucaine, hexylcaine, isobucaine,piperocaine, and a combination thereof. In some embodiments, the snakevenom or the lizard venom is selected from the group consisting ofnotexin, cardiotoxin, bungarotoxin, and a combination thereof. In someembodiments, the divalent cation is selected from the group consistingof Ba²⁺, Sr²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Ni²⁺, Co²⁺, a salt thereof, and acombination thereof.

In some embodiments, PGE2 compound is PGE2 and/or 16,16-dimethylprostaglandin E2 and the myotoxin is bupivacaine.

In some embodiments, PGE2 compound is PGE2 and/or 16,16-dimethylprostaglandin E2 and the myotoxin is bupivacaine. In some embodiments,the muscle condition is associated with muscle damage, injury, oratrophy.

Described herein is a pharmaceutical composition comprising thecomposition described herein and a pharmaceutically acceptable carrier.In some embodiments, the pharmaceutically acceptable carrier comprisesan aqueous base. In some embodiments, the pharmaceutically acceptablecarrier comprises a low viscosity compound. In some embodiments, the lowviscosity compound comprises gelatin. In some embodiments, the lowviscosity compound comprises a hydrogel.

Described herein is a method for promoting muscle regeneration and/orincreasing muscle mass in a subject in need thereof, the methodcomprising administering a combination of a PGE2 compound and a myotoxinto the subject. In some embodiments, the PGE2 compound is selected fromthe group consisting of PGE2, a PGE2 prodrug, a PGE2 receptor agonist, acompound that attenuates PGE2 catabolism, a compound that neutralizesPGE2 inhibition, a derivative thereof, an analog thereof, and acombination thereof. In some embodiments, the PGE2 derivative comprises16,16-dimethyl prostaglandin E2. In some embodiments, the compound thatattenuates PGE2 catabolism comprises a compound, neutralizing peptide,or neutralizing antibody that inactivates or blocks15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivates or blocksa prostaglandin transporter (PGT or SLCO2A1). In some embodiments, thePGE2 compound is PGE2.

In some embodiments, the myotoxin is selected from the group consistingof an anesthetic, a divalent cation, snake venom, lizard venom, beevenom, and a combination thereof. In some embodiments, the anesthetic isselected from the group consisting of an amino-amide anesthetic, anamino-ester anesthetic, and a combination thereof. In some embodiments,the amino-amide anesthetic is selected from the group consisting ofbupivacaine, levobupivacaine, articaine, ropivacaine, butanilicaine,carticaine, dibucaine, etidocaine, lidocaine, mepivacaine, prilocaine,trimecaine, and a combination thereof. In some embodiments, theamino-ester anesthetic is selected from the group consisting of anaminobenzoic acid ester anesthetic, a benzoic acid ester anesthetic, anda combination thereof. In some embodiments, the aminobenzoic acid esteranesthetic is selected from the group consisting of benzocaine,butacaine, butamben, chloroprocaine, dimethocaine, lucaine, meprylcaine,metabutethamine, metabutoxycaine, nitracaine, orthocaine, propoxycaine,procaine, proxymetacaine, risocaine, tetracaine, and a combinationthereof. In some embodiments, the benzoic acid ester anesthetic isselected from the group consisting of amylocaine, cocaine,cyclomethycaine, α-eucaine, β-eucaine, hexylcaine, isobucaine,piperocaine, and a combination thereof. In some embodiments, the snakevenom or the lizard venom is selected from the group consisting ofnotexin, cardiotoxin, bungarotoxin, and a combination thereof. In someembodiments, the divalent cation is selected from the group consistingof Ba²⁺, Sr²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Ni²⁺, Co²⁺, a salt thereof, and acombination thereof.

In some embodiments, PGE2 compound is PGE2 and/or 16,16-dimethylprostaglandin E2 and the myotoxin is bupivacaine. In some embodiments,the PGE2 compound and the myotoxin are administered concomitantly. Insome embodiments, the PGE2 compound and the myotoxin are administeredsequentially. In some embodiments, the PGE2 compound is administeredbefore the myotoxin. In some embodiments, the PGE2 compound isadministered after the myotoxin.

In some embodiments, administering the PGE2 compound, the myotoxin, orboth, comprises topical, oral, intraperitoneal, intramuscular,intra-arterial, intradermal, subcutaneous, intravenous, or intracardiacadministration. In some embodiments, administering comprisesintramuscular administration. In some embodiments, a dose of the PGE2compound, the myotoxin, or both, is determined based upon a targetmuscle size. In some embodiments, the target muscle is an abductorpollicis brevis muscle and the dose of the PGE2 compound, the myotoxin,or both, is about 10 μg.

In some embodiments, the method further comprises subjecting a targetmuscle to mechanical injury. In some embodiments, the mechanical injurycomprises cutting, burning, freezing, needle puncture, exercise, asurgical procedure, traumatic injury, or a combination thereof. In someembodiments, the method further comprises administering a population ofisolated muscle cells to the subject. In some embodiments, thepopulation of isolated muscle cells is autologous to the subject. Insome embodiments, the population of isolated muscle cells is allogeneicto the subject. In some embodiments, the population of isolated musclecells is purified. In some embodiments, population of isolated musclecells is cultured with the PGE2 compound, the myotoxin, or both, priorto being administered to the subject. In some embodiments, culturing thepopulation of isolated muscle cells with the PGE2 compound, themyotoxin, or both, comprises acute, intermittent, or continuous exposureof the population of isolated muscle cells to the PGE2 compound, themyotoxin, or both. In some embodiments, wherein administering thepopulation of isolated muscle cells comprises injecting or transplantingthe cells into the subject. In some embodiments, wherein administrationof the population of isolated muscle cells and administration of thePGE2 compound and the myotoxin are performed concomitantly. In someembodiments, administration of the population of isolated muscle cellsand administration of the PGE2 compound and the myotoxin are performedsequentially. In some embodiments, the subject has a muscle condition.

In some embodiments, the muscle condition is associated with muscledamage, injury, atrophy, or any combination thereof. In someembodiments, the muscle condition is selected from the group consistingof traumatic injury, acute muscle injury, acute nerve injury, chronicnerve injury, soft tissue hand injury, carpal tunnel syndrome (CTS),Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limbgirdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distalmuscular dystrophy (DD), inherited myopathies, myotonic musculardystrophy (MDD), mitochondrial myopathies, myotubular myopathy (MM),myasthenia gravis (MG), congestive heart failure, periodic paralysis,polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDScachexia, cardiac cachexia, stress induced urinary incontinence,sarcopenia, spinal muscular atrophy, fecal sphincter dysfunction, Bell'spalsy, rotator cuff injury, spinal cord injury, hip replacement, kneereplacement, wrist fracture, and diabetic neuropathy.

In some embodiments, the PGE2 compound and the myotoxin are administeredimmediately after the traumatic injury. In some embodiments, the subjectreceives a surgical procedure. In some embodiments, the surgicalprocedure is for the prevention of a nerve injury, reduction of a nerveinjury, repair of a nerve injury, or any combination thereof. In someembodiments, the surgical procedure comprises cutting a muscle,repairing a muscle, or both. In some embodiments, the subject receivesthe surgical procedure before administration of the PGE2 compound andthe myotoxin. In some embodiments, the subject receives the surgicalprocedure at the same time as administration of the PGE2 compound andthe myotoxin. In some embodiments, the subject receives the surgicalprocedure after administration of the PGE2 compound and the myotoxin. Insome embodiments, the nerve injury is a peripheral nerve injury. In someembodiments, the surgical procedure comprises a carpal tunnel releaseprocedure.

Described herein is a method for preventing or treating a musclecondition in a subject in need thereof, the method comprisingadministering a combination of a PGE2 compound and a myotoxin to thesubject. In some embodiments, the PGE2 compound is selected from thegroup consisting of PGE2, a PGE2 prodrug, a PGE2 receptor agonist, acompound that attenuates PGE2 catabolism, a compound that neutralizesPGE2 inhibition, a derivative thereof, an analog thereof, and acombination thereof. In some embodiments, the PGE2 derivative comprises16,16-dimethyl prostaglandin E2. In some embodiments, the compound thatattenuates PGE2 catabolism comprises a compound, neutralizing peptide,or neutralizing antibody that inactivates or blocks15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivates or blocksa prostaglandin transporter (PGT or SLCO2A1). In some embodiments,wherein the PGE2 compound is PGE2. In some embodiments, the myotoxin isselected from the group consisting of an anesthetic, a divalent cation,snake venom, lizard venom, bee venom, and a combination thereof. In someembodiments, the anesthetic is selected from the group consisting of anamino-amide anesthetic, an amino-ester anesthetic, and a combinationthereof. In some embodiments, the amino-amide anesthetic is selectedfrom the group consisting of bupivacaine, levobupivacaine, articaine,ropivacaine, butanilicaine, carticaine, dibucaine, etidocaine,lidocaine, mepivacaine, prilocaine, trimecaine, and a combinationthereof. In some embodiments, the amino-ester anesthetic is selectedfrom the group consisting of an aminobenzoic acid ester anesthetic, abenzoic acid ester anesthetic, and a combination thereof. In someembodiments, the aminobenzoic acid ester anesthetic is selected from thegroup consisting of benzocaine, butacaine, butamben, chloroprocaine,dimethocaine, lucaine, meprylcaine, metabutethamine, metabutoxycaine,nitracaine, orthocaine, propoxycaine, procaine, proxymetacaine,risocaine, tetracaine, and a combination thereof. In some embodiments,the benzoic acid ester anesthetic is selected from the group consistingof amylocaine, cocaine, cyclomethycaine, α-eucaine, β-eucaine,hexylcaine, isobucaine, piperocaine, and a combination thereof. In someembodiments, the snake venom or the lizard venom is selected from thegroup consisting of notexin, cardiotoxin, bungarotoxin, and acombination thereof. In some embodiments, the divalent cation isselected from the group consisting of Ba²⁺, Sr²⁺, Mg²⁺, Ca²⁺, Mn²⁺,Ni²⁺, Co²⁺, a salt thereof, and a combination thereof.

In some embodiments, the PGE2 compound is PGE2 and the myotoxin isbupivacaine. In some embodiments, the PGE2 compound and the myotoxin areadministered concomitantly. In some embodiments, the PGE2 compound andthe myotoxin are administered sequentially. In some embodiments, thePGE2 compound is administered before the myotoxin. In some embodiments,the PGE2 compound is administered after the myotoxin. In someembodiments, administering the PGE2 compound, the myotoxin, or both,comprises topical, oral, intraperitoneal, intramuscular, intra-arterial,intradermal, subcutaneous, intravenous, or intracardiac administration.In some embodiments, administering comprises intramuscularadministration. In some embodiments, a dose of the PGE2 compound, themyotoxin, or both, is determined based upon a target muscle size. Insome embodiments, the target muscle is an abductor pollicis brevismuscle and the dose of the PGE2 compound, the myotoxin, or both, isabout 10 μg.

In some embodiments, the method further comprises subjecting a targetmuscle to mechanical injury. In some embodiments, the mechanical injurycomprises cutting, burning, freezing, needle puncture, exercise, asurgical procedure, traumatic injury, or a combination thereof.

In some embodiments, the method further comprises administering apopulation of isolated muscle cells to the subject. In some embodiments,the population of isolated muscle cells is autologous to the subject. Insome embodiments, the population of isolated muscle cells is allogeneicto the subject. In some embodiments, the population of isolated musclecells is purified. In some embodiments, the population of isolatedmuscle cells is cultured with the PGE2 compound, the myotoxin, or both,prior to being administered to the subject. In some embodiments,culturing the population of isolated muscle cells with the PGE2compound, the myotoxin, or both, comprises acute, intermittent, orcontinuous exposure of the population of isolated muscle cells to thePGE2 compound, the myotoxin, or both. In some embodiments, thepopulation of isolated muscle cells comprises injecting or transplantingthe cells into the subject. In some embodiments, administration of thepopulation of isolated muscle cells and administration of the PGE2compound and the myotoxin are performed concomitantly. In someembodiments, administration of the population of isolated muscle cellsand administration of the PGE2 compound and the myotoxin are performedsequentially.

In some embodiments, the muscle condition is associated with muscledamage, injury, atrophy, or any combination thereof. In someembodiments, the muscle condition is selected from the group consistingof traumatic injury, acute muscle injury, acute nerve injury, chronicnerve injury, soft tissue hand injury, carpal tunnel syndrome (CTS),Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limbgirdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distalmuscular dystrophy (DD), inherited myopathies, myotonic musculardystrophy (MDD), mitochondrial myopathies, myotubular myopathy (MM),myasthenia gravis (MG), congestive heart failure, periodic paralysis,polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDScachexia, cardiac cachexia, stress induced urinary incontinence,sarcopenia, spinal muscular atrophy, fecal sphincter dysfunction, Bell'spalsy, rotator cuff injury, spinal cord injury, hip replacement, kneereplacement, wrist fracture, and diabetic neuropathy.

In some embodiments, the PGE2 receptor agonist and the myotoxin areadministered immediately after the traumatic injury. In someembodiments, the subject receives a surgical procedure. In someembodiments, the surgical procedure is for the prevention of a nerveinjury, reduction of a nerve injury, repair of a nerve injury, or anycombination thereof. In some embodiments, the surgical procedurecomprises cutting a muscle, repairing a muscle, or both. In someembodiments, the subject receives the surgical procedure beforeadministration of the PGE2 compound and the myotoxin. In someembodiments, the subject receives the surgical procedure at the sametime as administration of the PGE2 compound and the myotoxin. In someembodiments, the subject receives the surgical procedure afteradministration of the PGE2 compound and the myotoxin. In someembodiments, the nerve injury is a peripheral nerve injury. In someembodiments, the surgical procedure comprises a carpal tunnel releaseprocedure. In some embodiments, treating the subject results in animprovement in muscle strength, muscle coordination, or both, in thesubject.

Described herein is a method for preventing or treating a musclecondition in a subject in need thereof, the method comprisingadministering a prostaglandin E2 (PGE2) receptor agonist to the subject.In some embodiments, the PGE2 receptor agonist comprises a compound ofFormula (I), a derivative thereof, a pharmaceutically acceptable saltthereof, a solvate thereof, a stereoisomer thereof, or a combinationthereof,

-   -   wherein ring A is a substituted 4- to 6-membered cycloalkyl ring        or a substituted 4- to 6-membered cycloalkenyl ring that        comprises substituents R¹ and R² that are independently selected        from the group consisting of substituted C₁-C₁₀ alkyl and        substituted C₂-C₁₀ alkenyl, and ring A further comprises one or        more additional substituents.

In some embodiments, A is a substituted cyclopentyl ring or asubstituted cyclopentenyl ring. In some embodiments, the one or moreadditional substituents on ring A are selected from the group consistingof deuterium, hydroxy, amino, oxo, C₁-C₆ alkyl, and halogen. In someembodiments, the one or more additional substituents on ring A arehydroxy or oxo. In some embodiments, ring A has two additionalsubstituents that are taken together to form a covalent bond to form aheterocycloalkyl ring.

In some embodiments, ring A is selected from the group consisting of

In some embodiments, ring A is selected from the group consisting of

In some embodiments, ring A is

In some embodiments, R¹ is substituted C₁-C₁₀ alkyl.

In some embodiments, R¹ is substituted C₂-C₁₀ alkenyl.

In some embodiments, the substituent on R¹ is selected from the groupconsisting of deuterium, hydroxy, oxo, C₁-C₆ alkyl, —COOR³, and halogen,wherein R³ is hydrogen or C₁-C₆ alkyl.

In some embodiments, R¹ is selected from the group consisting of

In some embodiments, R¹ is selected from the group consisting of

In some embodiments, R¹ is

In some embodiments, R² is substituted C₁-C₁₀ alkyl. In someembodiments, R² is substituted C₂-C₁₀ alkenyl. In some embodiments, thesubstituent on R² is selected from the group consisting of deuterium,hydroxy, oxo, C₁-C₆ alkyl, —COOR³, and halogen, wherein R³ is hydrogenor C₁-C₆ alkyl. In some embodiments, R² is selected from the groupconsisting of

In some embodiments, R² is selected from the group consisting of

In some embodiments, R² is

In some embodiments, the compound of Formula (I), the pharmaceuticallyacceptable salt thereof, the solvate thereof, or the stereoisomerthereof is a compound of Formula (Ia), Formula (Ib), Formula (Ic), orFormula (Id), or a pharmaceutically acceptable salt, solvate, orstereoisomer thereof:

In some embodiments, the compound of Formula (I), the pharmaceuticallyacceptable salt thereof, the solvate thereof, or the stereoisomerthereof is a compound of Formula (Id), or a pharmaceutically acceptablesalt, solvate, or stereoisomer thereof.

In some embodiments, the PGE2 receptor agonist comprises PGE2,16,16-dimethyl prostaglandin E2, or both.

In some embodiments, the method further comprises administering amyotoxin to the subject. In some embodiments, the myotoxin is selectedfrom the group consisting of an anesthetic, a divalent cation, snakevenom, lizard venom, bee venom, and a combination thereof. In someembodiments, the anesthetic is selected from the group consisting of anamino-amide anesthetic, an amino-ester anesthetic, and a combinationthereof. In some embodiments, the amino-amide anesthetic is selectedfrom the group consisting of bupivacaine, levobupivacaine, articaine,ropivacaine, butanilicaine, carticaine, dibucaine, etidocaine,lidocaine, mepivacaine, prilocaine, trimecaine, and a combinationthereof. In some embodiments, the amino-ester anesthetic is selectedfrom the group consisting of an aminobenzoic acid ester anesthetic, abenzoic acid ester anesthetic, and a combination thereof. In someembodiments, the aminobenzoic acid ester anesthetic is selected from thegroup consisting of benzocaine, butacaine, butamben, chloroprocaine,dimethocaine, lucaine, meprylcaine, metabutethamine, metabutoxycaine,nitracaine, orthocaine, propoxycaine, procaine, proxymetacaine,risocaine, tetracaine, and a combination thereof. In some embodiments,the benzoic acid ester anesthetic is selected from the group consistingof amylocaine, cocaine, cyclomethycaine, α-eucaine, β-eucaine,hexylcaine, isobucaine, piperocaine, and a combination thereof. In someembodiments, the snake venom or the lizard venom is selected from thegroup consisting of notexin, cardiotoxin, bungarotoxin, and acombination thereof. In some embodiments, the divalent cation isselected from the group consisting of Ba²⁺, Sr²⁺, Mg²⁺, Ca²⁺, Mn²⁺,Ni²⁺, Co²⁺, a salt thereof, and a combination thereof. In someembodiments, the PGE2 receptor agonist is PGE2, 16,16-dimethylprostaglandin E2, or both, and the myotoxin is bupivacaine.

In some embodiments, the method further comprises subjecting a targetmuscle to mechanical injury. In some embodiments, the mechanical injurycomprises cutting, burning, freezing, needle puncture, exercise, asurgical procedure, traumatic injury, or a combination thereof.

In some embodiments, the method further comprises administering apopulation of isolated muscle cells to the subject. In some embodiments,the population of isolated muscle cells is autologous to the subject. Insome embodiments, the population of isolated muscle cells is allogeneicto the subject. In some embodiments, the population of isolated musclecells is purified. In some embodiments, the population of isolatedmuscle cells is cultured with the PGE2 receptor agonist prior to beingadministered to the subject. In some embodiments, culturing thepopulation of isolated muscle cells with the PGE2 compound comprisesacute, intermittent, or continuous exposure of the population ofisolated muscle cells to the PGE2 compound. In some embodiments,administering the population of isolated muscle cells comprisesinjecting or transplanting the cells into the subject. In someembodiments, administration of the population of isolated muscle cellsand administration of the PGE2 receptor agonist are performedconcomitantly. In some embodiments, administration of the population ofisolated muscle cells and administration of the PGE2 receptor agonistare performed sequentially.

In some embodiments, the muscle condition is associated with muscledamage, injury, atrophy, or any combination thereof. In someembodiments, the muscle condition is selected from the group consistingof traumatic injury, acute muscle, acute nerve injury, chronic nerveinjury, soft tissue hand injury, carpal tunnel syndrome (CTS), Duchennemuscular dystrophy (DMD), Becker muscular dystrophy, limb girdlemuscular dystrophy, amyotrophic lateral sclerosis (ALS), distal musculardystrophy (DD), inherited myopathies, myotonic muscular dystrophy (MDD),mitochondrial myopathies, myotubular myopathy (MM), myasthenia gravis(MG), congestive heart failure, periodic paralysis, polymyositis,rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiaccachexia, stress induced urinary incontinence, sarcopenia, spinalmuscular atrophy, fecal sphincter dysfunction, Bell's palsy, rotatorcuff injury, spinal cord injury, hip replacement, knee replacement,wrist fracture, and diabetic neuropathy.

In some embodiments, the PGE2 receptor agonist is administeredimmediately after the traumatic injury.

In some embodiments, the subject receives a surgical procedure. In someembodiments, the surgical procedure is for the prevention of a nerveinjury, reduction of a nerve injury, repair of a nerve injury, or anycombination thereof. In some embodiments, the surgical procedurecomprises cutting a muscle, repairing a muscle, or both. In someembodiments, the subject receives the surgical procedure beforeadministration of the PGE2 receptor agonist. In some embodiments, thesubject receives the surgical procedure at the same time asadministration of the PGE2 receptor agonist. In some embodiments, thesubject receives the surgical procedure after administration of the PGE2receptor agonist. In some embodiments, the nerve injury is a peripheralnerve injury. In some embodiments, the surgical procedure comprises acarpal tunnel release procedure. In some embodiments, no anesthetic isadministered to the subject.

Described herein is a kit for promoting muscle regeneration in a subjectin need thereof, increasing muscle mass in a subject in need thereof, orboth, or for preventing or treating a muscle condition in a subject inneed thereof, the kit comprising a composition described herein or apharmaceutical composition described herein. In some embodiments, thesubject has a muscle condition. In some embodiments, the musclecondition is associated with muscle damage, injury, atrophy, or anycombination thereof. In some embodiments, muscle condition is selectedfrom the group consisting of traumatic injury (e.g., acute muscletrauma, acute nerve trauma), acute muscle injury, acute nerve injury,chronic nerve injury, soft tissue hand injury, carpal tunnel syndrome(CTS), Duchenne muscular dystrophy (DMD), Becker muscular dystrophy,limb girdle muscular dystrophy, amyotrophic lateral sclerosis (ALS),distal muscular dystrophy (DD), inherited myopathies, myotonic musculardystrophy (MDD), mitochondrial myopathies, myotubular myopathy (MM),myasthenia gravis (MG), congestive heart failure, periodic paralysis,polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDScachexia, cardiac cachexia, stress induced urinary incontinence,sarcopenia, spinal muscular atrophy, fecal sphincter dysfunction, Bell'spalsy, rotator cuff injury, spinal cord injury, hip replacement, kneereplacement, wrist fracture, and diabetic neuropathy.

In some embodiments, the kit further comprises isolated muscle cells. Insome embodiments, the kit further comprises instructions for use. Insome embodiments, the kit further comprises one or more reagents. Insome embodiments, the kit further comprises a delivery device foradministering the composition, pharmaceutical composition, isolatedmuscle cells, or any combination thereof, to the subject.

Described herein is a method for treating a pelvic floor disorder in asubject in need thereof, the method comprising administering acombination of a PGE2 compound and a myotoxin to the subject. In someembodiments, the administering comprises administering the combinationof a PGE2 compound and a myotoxin to a pelvic floor muscle of thesubject. In some embodiments, the pelvic floor muscle is the levatorani, the coccygeus muscle, or both. In some embodiments, the levator anicomprises the pubococcygeus muscle, the iliococcygeus muscle, thepuborectalis muscle, or a combination thereof. In some embodiments, thepelvic floor disorder is selected from the group consisting of stressurinary incontinence, overactive bladder/urinary urgency incontinence,mixed urinary incontinence, pelvic organ prolapse, and fecalincontinence. In some embodiments, the method further comprisesadministering a therapy suitable to treat, prevent, or amelioratesymptoms associated with pelvic floor disorders to the subject. In someembodiments, the additional therapy is selected from the groupconsisting of muscle training/biofeedback, neuromodulation,pharmacotherapy, surgery, and a combination thereof.

Described herein is a method for treating an ocular disease or disorderin a subject in need thereof, the method comprising administering acombination of a PGE2 compound and a myotoxin to the subject. In someembodiments, the ocular disease or disorder comprises impaired eyelidfunction. In some embodiments, the administering comprises administeringthe combination of a PGE2 compound and a myotoxin to an eyelid muscle ofthe subject. In some embodiments, the eyelid muscle is selected from thegroup consisting of Muller's muscle, ocipitofrontalis muscle,temporoparietalis muscle, procerus muscle, nasalis muscle, depressorsepti nasi muscle, orbicularis oculi muscle, corrugator superciliimuscle, depressor supercilii muscle, anterior auricular muscles,superior auricular muscle, posterior auricular muscle, orbicularis orismuscle, depressor anguli oris muscle, risorius, zygomaticus majormuscle, zygomaticus minor muscle, levator labii superioris, levatorlabii superioris alaeque nasi muscle, depressor labii inferioris muscle,levator anguli oris, buccinator muscle, mentalis, frontalis muscle, anda combination thereof. In some embodiments, the impaired eyelid functionis selected from the group consisting of eyelid drooping, ptosis,dermatochalasis, and a combination thereof. In some embodiments, themethod further comprises, prior to, during, or after the administering,performing eyelift surgery on the subject. In some embodiments, theimpaired eyelid function is associated with irregular astigmatism. Insome embodiments, the ocular disease or disorder is selected from thegroup consisting of impaired blinking, wet eye syndrome, dry eyesyndrome, lacrimal gland atrophy, 7th facial nerve palsy, recurringstyes, and a combination thereof. In some embodiments, the administeringcomprises administering the combination of a PGE2 compound and amyotoxin to an eye muscle of the subject. In some embodiments, the eyemuscle is selected from the group consisting of muscle of Riolan,Horner's muscle, frontalis muscle, ocipitofrontalis muscle,temporoparietalis muscle, procerus muscle, nasalis muscle, depressorsepti nasi muscle, orbicularis oculi muscle, corrugator superciliimuscle, depressor supercilii muscle, anterior auricular muscles,superior auricular muscle, posterior auricular muscle, orbicularis orismuscle, depressor anguli oris muscle, risorius, zygomaticus majormuscle, zygomaticus minor muscle, levator labii superioris, levatorlabii superioris alaeque nasi muscle, depressor labii inferioris muscle,levator anguli oris, buccinator muscle, mentalis, and a combinationthereof. In some embodiments, the ocular disease or disorder isectropion or entropion. In some embodiments, the administering comprisesadministering the combination of a PGE2 compound and a myotoxin to aneye muscle of the subject. In some embodiments, the eye muscle isselected from the group consisting of frontalis muscle, ocipitofrontalismuscle, temporoparietalis muscle, procerus muscle, nasalis muscle,depressor septi nasi muscle, orbicularis oculi muscle, corrugatorsupercilii muscle, depressor supercilii muscle, anterior auricularmuscles, superior auricular muscle, posterior auricular muscle,orbicularis oris muscle, depressor anguli oris muscle, risorius,zygomaticus major muscle, zygomaticus minor muscle, levator labiisuperioris, levator labii superioris alaeque nasi muscle, depressorlabii inferioris muscle, levator anguli oris, buccinator muscle,mentalis, and a combination thereof. In some embodiments, the methodfurther comprises, prior to, during, or after the administering,performing eyelid surgery on the subject. In some embodiments, theeyelid surgery is a lateral tarsal strip procedure. In some embodiments,the ocular disease or disorder is strabismus or nystagmus. In someembodiments, the administering comprises administering the combinationof a PGE2 compound and a myotoxin to an extraocular muscle of thesubject. In some embodiments, the extraocular muscle is selected fromthe group consisting of lateral rectus, medial rectus, superior rectus,inferior rectus, superior oblique, inferior oblique, and a combinationthereof. In some embodiments, the strabismus is associated with any oneof the following Apert syndrome, cerebral palsy, congenital rubella,hemangioma, Incontinentia Pigmenti, Noonan syndrome, Prader-Willisyndrome, retinopathy of prematurity, retinoblastoma, traumatic braininjury, trisomy-18, botulism, diabetes mellitus, Graves' disease,Guillain-Barre syndrome, injury to an eye, shellfish poisoning, stroke,and vision loss from an eye disease or injury. In some embodiments, thenystagmus is associated with any one of the following infantilenystagmus syndrome, intake of drugs or medications, excessive alcoholconsumption, sedating medicine that impairs a function of the labyrinth,head injury, an inner ear disorder, stroke, thiamine or vitamin B12deficiency, and Parkinson's disease. In some embodiments, the methodfurther comprises, prior to, during, or after the administering,performing eye surgery on the subject. In some embodiments, the oculardisease or disorder is associated with impaired iris function. In someembodiments, the administering comprises administering the combinationof a PGE2 compound and a myotoxin to an iris sphincter muscle or an irisdilator muscle of the subject. In some embodiments, the ocular diseaseor disorder is presbyopia. In some embodiments, the administeringcomprises administering the combination of a PGE2 compound and amyotoxin to a ciliary muscle of the subject. In some embodiments, theocular disease or disorder is myopia. In some embodiments, theadministering comprises administering the combination of a PGE2 compoundand a myotoxin to a ciliary muscle, a muscle in the sclera, a musclearound the sclera, an intraocular muscle, or a combination thereof, ofthe subject.

Described herein is a method for treating a musculoskeletal disorder ofa subject in need thereof, the method comprising administering acombination of a PGE2 compound and a myotoxin to the subject. In someembodiments, the musculoskeletal disorder comprises impaired handfunction. In some embodiments, the administering comprises administeringthe combination of a PGE2 compound and a myotoxin to a hand muscle ofthe subject. In some embodiments, the hand muscle is selected from thegroup consisting of abductor pollicis brevis, flexor pollicis brevis,opponens pollicis, abductor digiti minimi, flexor digiti minimi brevis,opponens digiti minimi, a dorsal interossei muscle, a volar interosseimuscle, a lumbrical muscle, palmaris brevis, adductor pollicis, abductorpollicis longus, extensor pllicis brevis, flexor pollicis longus, flexorcarpi radialis, flexor digitorum profundus, flexor digitorumsuperficialis, flexor carpi ulnaris, extensor carpi radialis longus,extensor carpi radialis brevis, extensor indicis, extensor digitorumcommunis, extensor digiti minimi, extensor carpi ulnaris, and acombination thereof.

In some embodiments, the method further comprises, prior to, during, orafter the administering, performing hand surgery on the subject. In someembodiments, the musculoskeletal disorder comprises impaired thumbfunction. In some embodiments, the administering comprises administeringthe combination of a PGE2 compound and a myotoxin to a hand muscle ofthe subject. In some embodiments, the hand muscle is selected from thegroup consisting of abductor pollicis brevis, opponens pollicis, flexorpollicis brevis, and a combination thereof. In some embodiments, theimpaired thumb function is due to thenar atrophy.

In some embodiments, the method further comprises, prior to, during, orafter the administering, performing hand surgery on the subject. In someembodiments, the hand surgery is carpal tunnel syndrome surgery. In someembodiments, the impaired thumb function is associated with cubitaltunnel syndrome or thoracic outlet syndrome.

In some embodiments, the musculoskeletal disorder comprises impairedfoot function. In some embodiments, the administering comprisesadministering the combination of a PGE2 compound and a myotoxin to afoot muscle of the subject. In some embodiments, the foot muscle isselected from the group consisting of flexor digitorum brevis, abductorhallucis, abductor digiti minimi, quadratus plantae, lumbricals, flexordigitorum longus, adductor hallucis, flexor hallucis brevis, flexorhallucis longus, flexor digiti minimi brevis, dorsal interossei, plantarinterossei, flexor hallucis medialis, flexor hallucis brevis lateralis,adductor hallucis transverse, adductor hallucis oblique, and acombination thereof. In some embodiments, the impaired foot function isdue to plantar fasciitis. In some embodiments, the impaired footfunction is foot drop. In some embodiments, the administering comprisesadministering the combination of a PGE2 compound and a myotoxin to afoot muscle or a lower leg muscle of the subject. In some embodiments,the foot muscle or lower leg muscle is selected from the groupconsisting of anterior tibialis muscle, fibularis tertius, extensordigitorum longus, extensor hallucis longus, and a combination thereof.

In some embodiments, the method further comprises, prior to, during, orafter the administering, performing surgery on the subject. In someembodiments, the foot drop is associated with any one of the following:compression of a peroneal nerve; a nerve root injury; musculardystrophy; amyotrophic lateral sclerosis; multiple sclerosis; or stroke.In some embodiments, the musculoskeletal disorder is disuse-inducedmuscle atrophy. In some embodiments, the disuse-induced muscle atrophyis caused by a distal radius fracture. In some embodiments, theadministering comprises administering the combination of a PGE2 compoundand a myotoxin to a hand muscle or lower arm muscle of the subject. Insome embodiments, the hand muscle or lower arm muscle is selected fromthe group consisting of flexor carpi radialis, flexor pollicis longus,flexor digitorum superficialis, flexor digitorum profundus, flexor carpiulnaris, extensor carpi radialis brevis, extensor carpi radialis longus,extensor pollicis longus, extensor digitorum communis, extensor carpiulnaris, and a combination thereof.

In some embodiments, the method further comprises prior to, during, orafter the administering, performing surgery on the subject. In someembodiments, the surgery is wrist arthroscopy. In some embodiments, thedisuse-induced muscle atrophy is caused by a hip fracture. In someembodiments, the administering comprises administering the combinationof a PGE2 compounds and a myotoxin to a hip muscle of the subject. Insome embodiments, the hip muscles is selected from the group consistingof iliacus, psoas major, gluteus maximus, gluteus medius, gluteusminimus, tensor fasciae latae, superior gemellus, inferior gemellus,obturator internus, obturator externus, quadratus femoris, piriformis,adductor magnus, adductor longus, adductor brevis, adductor minimus,pectineus, rectus femoris, vastus lateralis, vastus medialis, vastusintermedius, quadriceps femoris, Sartorius, biceps femoris,semitendinosus, semimembranosus, psoas minor, iliopsoas, gracilis, and acombination thereof.

In some embodiments, the method further comprises, prior to, during, orafter the administering, performing surgery on the subject. In someembodiments, the surgery is joint arthroplasty. In some embodiments, thedisuse-induced muscle atrophy is caused by a rotator cuff injury. Insome embodiments, the administering comprises administering thecombination of a PGE2 compound and a myotoxin to a rotator cuff muscleof the subject. In some embodiments, the rotator cuff muscle is selectedfrom the group consisting of supraspinatus, infraspinatus,subscapularis, teres minor, and a combination thereof.

Described herein is a method for treating gastroesophageal refluxdisease (GERD) in a subject in need thereof, the method comprisingadministering a combination of a PGE2 compound and a myotoxin to thesubject. In some embodiments, the administering comprises administeringthe combination of a PGE2 compounds and a myotoxin to a crural diaphragmof the subject.

Described herein is a method for treating obstructive sleep apnea in asubject in need thereof, the method comprising administering acombination of a PGE2 compound and a myotoxin to the subject. In someembodiments, the administering comprises administering the combinationof a PGE2 compound and a myotoxin to an upper airway muscle of thesubject. In some embodiments, the upper airway muscle is selected fromthe group consisting of genioglossus, tensor palatine, a geniohyoidmuscle, and a combination thereof.

Described herein is a method for treating oculopharyngeal musculardystrophy in a subject in need thereof, the method comprisingadministering a combination of a PGE2 compound and a myotoxin to thesubject. In some embodiments, the administering comprises administeringthe combination of a PGE2 compound and a myotoxin to a muscle of theupper eyelid or a muscle of the throat.

Described herein is a method for treating diabetic neuropathy in asubject in need thereof, the method comprising administering acombination of a PGE2 compound and a myotoxin to the subject. In someembodiments, the administering comprises administering the combinationof a PGE2 compound and a myotoxin to a small muscle of a foot, a lowerleg muscle, or an intrinsic muscle of a foot. In some embodiments, thePGE2 compound is selected from the group consisting of PGE2, a PGE2prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2catabolism, a compound that neutralizes PGE2 inhibition, a derivativethereof, an analog thereof, and a combination thereof. In someembodiments, the PGE2 derivative comprises 16,16-dimethyl prostaglandinE2. In some embodiments, the compound that attenuates PGE2 catabolismcomprises a compound, neutralizing peptide, or neutralizing antibodythat inactivates or blocks 15-hydroxyprostaglandin dehydrogenase(15-PGDH) or inactivates or blocks a prostaglandin transporter (PGT orSLCO2A1). In some embodiments, the PGE2 compound is PGE2. In someembodiments, the myotoxin is selected from the group consisting of ananesthetic, a divalent cation, snake venom, lizard venom, bee venom, anda combination thereof. In some embodiments, the anesthetic is selectedfrom the group consisting of an amino-amide anesthetic, an amino-esteranesthetic, and a combination thereof. In some embodiments, theamino-amide anesthetic is selected from the group consisting ofbupivacaine, levobupivacaine, articaine, ropivacaine, butanilicaine,carticaine, dibucaine, etidocaine, lidocaine, mepivacaine, prilocaine,trimecaine, and a combination thereof. In some embodiments, theamino-ester anesthetic is selected from the group consisting of anaminobenzoic acid ester anesthetic, a benzoic acid ester anesthetic, anda combination thereof. In some embodiments, the aminobenzoic acid esteranesthetic is selected from the group consisting of benzocaine,butacaine, butamben, chloroprocaine, dimethocaine, lucaine, meprylcaine,metabutethamine, metabutoxycaine, nitracaine, orthocaine, propoxycaine,procaine, proxymetacaine, risocaine, tetracaine, and a combinationthereof. In some embodiments, the benzoic acid ester anesthetic isselected from the group consisting of amylocaine, cocaine,cyclomethycaine, α-eucaine, β-eucaine, hexylcaine, isobucaine,piperocaine, and a combination thereof. In some embodiments, the snakevenom or the lizard venom is selected from the group consisting ofnotexin, cardiotoxin, bungarotoxin, and a combination thereof. In someembodiments, the divalent cation is selected from the group consistingof Ba²⁺, Sr²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Ni²⁺, Co²⁺, a salt thereof, and acombination thereof. In some embodiments, the PGE2 compound is PGE2and/or 16,16-dimethyl prostaglandin E2 and the myotoxin is bupivacaine.In some embodiments, the PGE2 compound and the myotoxin are administeredconcomitantly. In some embodiments, the PGE2 compound and the myotoxinare administered sequentially. In some embodiments, the PGE2 compound isadministered before the myotoxin. In some embodiments, the PGE2 compoundis administered after the myotoxin. In some embodiments, administeringthe PGE2 compound, the myotoxin, or both, comprises topical, oral,intraperitoneal, intramuscular, intra-arterial, intradermal,subcutaneous, intravenous, or intracardiac administration. In someembodiments, administering comprises intramuscular administration. Insome embodiments, wherein a dose of the PGE2 compound, the myotoxin, orboth, is determined based upon a target muscle size. In someembodiments, the target muscle is an abductor pollicis brevis muscle andthe dose of the PGE2 compound, the myotoxin, or both, is about 10 μg.

In some embodiments, the method further comprises subjecting a targetmuscle to mechanical injury. In some embodiments, the mechanical injurycomprises cutting, burning, freezing, needle puncture, exercise, asurgical procedure, traumatic injury, or a combination thereof. In someembodiments, the method further comprises administering a population ofisolated muscle cells to the subject. In some embodiments, thepopulation of isolated muscle cells is autologous to the subject. Insome embodiments, the population of isolated muscle cells is allogeneicto the subject. In some embodiments, the population of isolated musclecells is purified. In some embodiments, the population of isolatedmuscle cells is cultured with the PGE2 compound, the myotoxin, or both,prior to being administered to the subject. In some embodiments,culturing the population of isolated muscle cells with the PGE2compound, the myotoxin, or both, comprises acute, intermittent, orcontinuous exposure of the population of isolated muscle cells to thePGE2 compound, the myotoxin, or both. In some embodiments, administeringthe population of isolated muscle cells comprises injecting ortransplanting the cells into the subject. In some embodiments,administration of the population of isolated muscle cells andadministration of the PGE2 compound and the myotoxin are performedconcomitantly. In some embodiments, administration of the population ofisolated muscle cells and administration of the PGE2 compound and themyotoxin are performed sequentially.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an illustration of the abductor pollicis brevis (APB)muscle (left). The arrows mark the locations of APB atrophy, also shownin the photo on the right.

FIGS. 2A-2H show that transient PGE2 treatment promotes young MuSCproliferation in vitro. FIG. 2A: PGE2 levels after young tibialisanterior (TA) muscle injury (notexin, NTX); controls are uninjuredcontralateral TAs assayed by ELISA; (n=4 mice per time point). FIG. 2B:Expression of PGE2 synthesizing enzymes (Ptges2 and Ptges) by MuSCsafter notexin injury by RT-qPCR, (n=3 mice per time point). FIG. 2C:Increase in MuSC numbers after 24 hr treatment with vehicle (−) or PGE2(10 ng/ml), and subsequent culture on hydrogel until day 7 (acutetreatment); (n=12 mice in 4 independent experiments). FIG. 2D: Increasein MuSC numbers after transient 24 hr treatment with vehicle (−) or PGE2(10 ng/ml) in absence or presence of EP4 antagonist (ONO-AE3-208, 1 μM);(n=9 mice assayed in 3 independent experiments). FIGS. 2E-2G:Proliferation of EP4 null MuSCs. EP4^(f/f) (null) MuSCs were transducedwith a lentiviral vector encoding GFP/luciferase and treated withlentiviral vector encoding Cre (+Cre) or without (−Cre; empty vector) todelete EP4 alelles. Subsequently MuSCs were treated with vehicle (−) orPGE2 (10 ng/ml) for 24 hr and cultured on hydrogels for three days. FIG.2E: Scheme depicting EP4-null MuSC analysis. FIG. 2F: EP4 null MuSCnumbers; (n=6 mice in 2 independent experiments). FIG. 2G:Representative image. Bar=40 μm; GFP, green; mCherry, red. FIG. 2H: MuSCnumbers after culture in charcoal stripped medium treated with vehicle(−) or PGE2 (10 ng/ml) every two days for 7 days on hydrogels; (n=3 micewith 3 technical replicates). *P<0.05, **P<0.001, ***P<0.0005****P<0.0001. ANOVA test with Bonferroni correction for multiplecomparisons (FIGS. 2A, 2B, 2D, and 2F); paired t-test (FIG. 2C);Mann-Whitney test (FIG. 2H). Means+s.e.m. n.s., non-significant.

FIGS. 3A-3J show an aberrant response of aged MuSCs to PGE2. FIG. 3A:PGE2 levels after aged TA injury (notexin, NTX); controls are uninjuredcontralateral TAs assayed by ELISA; (n=4 mice per time point). FIG. 3B:PGE2 levels in TAs of uninjured young (n=7 mice) and aged (n=5 mice)mice assayed by ELISA. FIG. 3C: Scheme showing PGE2 catabolism viadegrading enzyme 15-PGDH to its inactive PGE metabolite,13,14-dihydro-15-keto PGE2 (PGEM). FIG. 3D: Levels of PGEM quantified bymass spectrometry; (n=4 mice per age group). FIG. 3E: Expression of PGE2degrading enzyme 15-PGDH (Hpgd); (n=3 mice with 2 technical replicates).FIG. 3F: Increase in aged MuSC numbers after acute 24 hr treatment withvehicle (−), PGE2 (10 ng/ml) or the 15-PGDH inhibitor, SW033291 (1 μM;SW) assayed at day 7; (n=15 mice in 5 independent experiments). FIG. 3G:Aged MuSC numbers after culture in charcoal stripped medium treated withvehicle (−) or PGE2 (10 ng/ml) every two days for 7 days on hydrogels;(n=3 mice with 3 technical replicates). FIG. 3H: Scheme depicting PGE2effects on MuSCs. PGE2 acts through the EP4 receptor/cAMP (cyclic AMP)signaling pathway to promote proliferation. In aged MuSCs, followingintracellular transport by PGT (prostaglandin transporter), PGE2catabolism is mediated by 15-PGDH to the inactive form, PGEM. FIG. 3I:Trajectories from a clone of aged MuSCs tracked by time-lapse microscopyfor 48 h in a microwell for control (left) and after acute treatmentwith PGE2 (right). The trajectory of the original cell and each of itsnewborn progeny are represented by a different color. FIG. 3J: Change inaged MuSC live cell counts (numbers) in clones tracked by time-lapsemicroscopy for control (left, n=32 clones) and after acute treatmentwith PGE2 (right, n=45 clones). The proportion of live cells in eachgeneration (G1-G6) at all timepoints is shown as cell number normalizedto a starting population of 100 single MuSCs. The percent increase inlive cell count was 4.0% (control) and 5.4% (PGE2-treated) (top panels).Change in aged MuSC dead cell counts (numbers) in clones tracked bytime-lapse microscopy for control (left) and after acute treatment withPGE2 (right). The proportion of dead cells in each generation (G1-G6) atall timepoints is shown as cell number normalized to a startingpopulation of 100 single MuSCs. The percent increase in dead cell countwas 1.0% (control) and 0.1% (PGE2-treated) (bottom panels). *P<0.05,**P<0.001, ***P<0.0005. ANOVA test with Bonferroni correction formultiple comparisons (FIGS. 3A and 3F); Mann-Whitney test (FIGS. 3B, 3D,3E, and 3G). Means±s.e.m. n. s., non-significant.

FIGS. 4A-4D show that acute PGE2 treatment promotes MuSC engraftment andregeneration in vivo. FIG. 4A: Engraftment of cultured GFP/luc-labeledyoung MuSCs (250 cells) isolated from transgenic mice after acutetreatment with vehicle (−) or PGE2 as described in FIG. 2C. Transplantscheme (top). Non-invasive bioluminescence imaging (BLI) signal measuredas radiance for each TA; (n=5 mice per condition) (bottom). FIG. 4B:Engraftment of GFP/luc-labeled EP4^(f/f) MuSCs (1,000 cells) treatedwith Cre (+Cre) or without (−Cre; empty vector) in culture to delete EP4 alelles. EP4^(f/f) MuSCs were transduced with a lentiviral vectorencoding GFP/luciferase for BLI. Transplant scheme (top). BLI signalspost-transplant (n=5 mice per condition (bottom). FIG. 4C: Engraftmentof freshly sorted GFP/luc-labeled young MuSCs (250 cells) coinjectedwith vehicle (−) or dmPGE2. Transplant scheme (top). BLI signalspost-transplant; (n=4 and n=5 mice for vehicle and dmPGE2 treated,respectively). FIG. 4D: Engraftment of GFP/luc-labeled aged MuSCs (250cells) coinjected with vehicle (−) or dmPGE2; (n=3 mice per condition)(bottom). Aged MuSCs were transduced with a lentiviral vector encodingGFP/luciferase for BLI. Transplant scheme (top). BLI signalspost-transplant expressed as average radiance (p s⁻¹ cm⁻² sr⁻¹).Representative BLI images for each condition. Bar=5 mm (FIGS. 4A-4D).Data are representative of two independent experiments. *P<0.05,**P<0.001 and ***P<0.0005. ANOVA test for group comparisons andsignificant difference for endpoints by Fisher's test. Means+s.e.m.

FIGS. 5A-5R show that intramuscular injection of PGE2 alone promotesMuSC expansion, improves regeneration, and increases force. Young:(FIGS. 5A-5D) TA muscles of young mice were injected with vehicle (−) ordmPGE2 48 hr post-cardiotoxin (CTX) injury; (n=3 mice per condition).FIG. 5A: Scheme of experimental procedure (top). Representative TAcross-section (bottom) with nuclei (DAPI; blue), LAMININ (green) andPAX7 (red) staining 14 days after cardiotoxin injury. Arrowheadsindicate PAX7⁺ MuSCs. Bar=40 μm. FIG. 5B: Increase in endogenous MuSCsby immunofluorescence of PAX7 expressing satellite cells per 100 fibersin cross-sections of TAs from young mice. FIG. 5C: Myofibercross-sectional areas (CSA) in vehicle (−, open white bar) and dmPGE2treated (filled blue bar) young TAs quantified using the BaxterAlgorithms for Myofiber Analysis. FIG. 5D: Distribution of small (<1,000μm² CSA) and large (>1,000 μm² CSA) myofibers. (FIGS. 5E-5G) Increase inendogenous MuSCs assayed by Pax7-luciferase.Pax7^(CreERT2);Rosa26-LSL-Luc mice were treated intraperitoneally withtamoxifen (TAM), TAs subjected to cardiotoxin (CTX) injury, injectedwith vehicle (−) or dmPGE2 3 days later and monitored by BLI; (n=3 miceper condition). FIG. 5E: Scheme of experimental procedure. FIG. 5F: BLI(n=3 mice per condition). FIG. 5G: Representative BLI image. Bar=5 mm.Aged: (FIGS. 5H-5K) TAs of aged mice were treated in vivo with vehicle(−) or dmPGE2 treatment 48 hr post-cardiotoxin (CTX) injury; (n=3 miceper condition). FIG. 5H: Scheme of experimental procedure (top).Representative TA cross-section (bottom) with nuclei (DAPI;blue),LAMININ (green) and PAX7 (red) staining 14 days after cardiotoxininjury. Arrowheads indicate PAX7⁺ muscle stem cells. Bar=40 μm. FIG. 5I:Increase in endogenous MuSCs as in FIG. 5B for aged mice. FIG. 5J:Myofiber cross-sectional area (CSA) as in FIG. 5C for aged TAs. FIG. 5K:Distribution of CSA as in FIG. 5D for aged TAs. (FIGS. 5L-5P) Increasein strength in aged mice measured in vivo as muscle contractile forceafter downhill treadmill run. Mice were subject to a 20° downhilltreadmill run for 2 consecutive weeks and force was assayed at week 5.During the first week, medial and lateral gastrocnemius (GA) of agedmice were injected either with vehicle (−) or dmPGE2. n=10 or 8biological replicates for vehicle (−) treated or dmPGE2 treated,respectively, with 5 technical replicates each. FIG. 5L: Experimentalscheme. Representative twitch force (FIG. 5M) and tetanic force (FIG.5N). Specific muscle twitch forces (FIG. 5O) and specific muscle tetanicforce (FIG. 5P) were calculated by normalizing force to physiologicalcross sectional areas (PCSA). Paired t-test (FIGS. 5B, 5D, 5I and 5K);ANOVA test for group comparison and significant difference for theendpoint by Fisher's test (FIG. 5F); Mann-Whitney test (FIGS. 5O and5P). *P<0.05, **P<0.001 and ****P<0.0001. Means+s.e.m. FIG. 5Q: Muscletwitch forces in aged mice that were administered PGE2 or vehicle only.FIG. 5R: Muscle tetanic force in mice that were administered PGE2 orvehicle only.

FIGS. 6A-6K show that PGE2 promotes MuSC expansion. FIG. 6A: PGE2 levelsday 3 after cryoinjury for tibialis anterior (TA) hindlimb muscles ofyoung mice compared to contralateral uninjured controls as assayed byELISA; (n=4 mice per time point per condition). FIG. 6B: Representativeimage of dividing muscle stem cells (MuSCs) labelled with EdU (red)during 1 hr after treatment with PGE2 (10 ng/ml) for 24 h (d0 to d1) orvehicle (−), and stained for MYOGENIN (green). Bar represents 40 μm.FIG. 6C: Percentage of dividing MuSCs labeled with EDU as in (b); (n=6mice with 3 technical replicates in two independent experiments). FIG.6D: Increase in proliferation measured by the metabolic viability assayVisionBlue after treatment with vehicle (−) or indicated doses of PGE2(1-200 ng/ml); (n=6 mice with 3 technical replicates in two independentexperiments). FIG. 6E: Expression of prostaglandin receptors (Ptger 1-4)by MuSCs after 24 hr treatment with vehicle (−) or PGE2; (n=3 mice with2 technical replicates). FIG. 6F: Increase in cAMP levels in MuSCs after1 hr PGE2 treatment relative to untreated controls (−); (n=6 mice with 3technical replicates assayed in 2 independent experiments). FIGS. 6G-6H:Expression of Pax7 (FIG. 6G) and Myogenin (FIG. 6H) by MuSCs after 24 hrtreatment with vehicle (−) or PGE2; (n=3 mice with 2 technicalreplicates). FIGS. 6I-6J: EP4^(f/f) MuSCs were transduced with alentiviral vector encoding GFP/luciferase and treated with lentiviralvector encoding Cre (+Cre) or without (−Cre; empty vector) to delete EP4alelles. Bar graphs show percentage of +Cre MuSCs (FIG. 6I) and GFP/Luc⁺MuSCs (FIG. 6J). FIG. 6K: Representative image of MuSCs in hydrogelculture after 7 days in myoblast medium containing charcoal strippedfetal bovine supplemented with vehicle (−) or PGE2 (10 ng/ml) every twodays. Bar represents 40 μm. *P<0.05, **P<0.001, ***P<0.0005. Pairedt-test (FIGS. 6A, 6E, 6G, and 6H); Mann-Whitney test (FIG. 6C).Means+s.e.m. n.s., non-significant.

FIGS. 7A-7C show mass spectrometry analysis of young and aged muscle todetect prostaglandins and PGE2 metabolites. FIG. 7A: Chemicalstructures, chemical formula, exact mass and molecular weight ofanalyzed prostaglandins (PGE2, PGF2α and PGD2) and PGE2 metabolites(15-keto PGE2 and 13,14-dihydro-15-keto PGE2). The internal standardsPGF2α-D9 and PGE2-D9 were added to all composite standards. FIG. 7B:Calibration lines for liquid chromatography-electrosprayionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis wereprepared by diluting stock solutions to final concentrations of 0.1ng/ml to 500 ng/ml. Standard curve equations and correlationcoefficients are shown for each standard. FIG. 7C: Representativechromatogram. The separate peaks show excellent chromatographicresolution of the analyzed prostaglandins and their metabolites. cps,counts per second.

FIGS. 8A-8G show that aged MuSCs increase proliferation and cellsurvival in response to PGE2 treatment. FIGS. 8A-8C: mRNA levelsmeasured by qRT-PCR were normalized to Gapdh for young and aged MuSCs;(n=3 mice with 2 technical replicates). FIG. 8A: Prostaglandintransporter (PGT) encoded by the Slco2a1 gene. FIG. 8B: PGE2synthesizing enzymes, Ptges and Ptges2. FIG. 8C: EP1-4 receptors encodedby the genes Ptger 1-4. FIG. 8D: Pax7 mRNA levels in MuSCs after 24 hrtreatment with vehicle (−) or PGE2 treatment; (n=3 mice with 2 technicalreplicates). FIG. 8E: Single aged MuSC clones tracked by time-lapsemicroscopy after acute treatment with vehicle (−; top) or PGE2 (bottom).For each clone the resulting number of live (open bar) and dead (blackbar) cells after 48 h timelapse tracking is shown. FIG. 8F:Proliferation curve of tracked live aged MuSCs assessed by time-lapsemicroscopy for vehicle (−) or transient PGE2 treatment during 48 h. FIG.8G: Flow cytometry analysis of apoptotic Annexin V⁺ on aged MuSCs after24 hr treatment with vehicle (−) or PGE2 and analyzed 7 days later aftergrowth on hydrogels; (n=9 mice in 3 independent experiments).Mann-Whitney test (FIGS. 8A-8D) and paired t-test (FIG. 8G) at α=0.05.Means+s.e.m. n.s., non-significant.

FIGS. 9A-9B show Baxter Algorithms for Myofiber Analysis of musclecross-sectional area. FIG. 9A: Representative cross-sectional images oftibialis anterior myofibers of young mice treated in vivo with vehicle(−) or PGE2 48 hr post-cardiotoxin (CTX) injury. Images show stainingwith LAMININ, green and DAPI, blue. FIG. 9B: The correspondingsegmentation images from FIG. 9A analyzed by the Baxter Algorithms forMyofiber Analysis to determine the cross sectional area (CSA) oftransverse sections of myofibers (bottom) at day 14 post-injury. Barrepresents 40 μm.

FIGS. 10A-10G show that deletion of PGE2 receptor EP4 in MuSCs decreasesregeneration and force of skeletal muscle after injury. Tibialisanteriors (TAs) of Pax7-specific EP4 conditional knockout mice(Pax7^(CreERT2);EP4^(fl/fl)) treated with tamoxifen were assayed at 6(FIGS. 10C-10D), 21 (FIGS. 10B and 10E), and 14 (FIGS. 10F and 10G) dayspost-notexin injury; (n=3 mice per condition). FIG. 10A: Experimentalscheme. FIG. 10B: Expression of Ptger4 (EP4 receptor) in sorted MuSCs(α⁷⁺ CD34⁺ lin⁻) from control or EP4 KO mice post-injury. FIG. 10C:Representative TA cross-section. DAPI, blue; Embryonic Myosin HeavyChain (eMyHC), red. Bar=40 μm. FIG. 10D: Percentage of eMyHC+fibers.FIG. 10E: Myofiber cross-sectional areas (CSA) in control andPax7-specific EP4 knockout TAs. FIG. 10F: Muscle twitch forces and (FIG.10G) muscle tetanic force at day 14 post-notexin injury. Mann-Whitneytest (FIGS. 10B, 10C, 10F, and 10G); ANOVA test for group comparison andsignificant difference for each bin by Fisher's test (FIG. 10E).*P<0.05, ***P<0.0005, and ****P<0.0001. Means+s.e.m.

FIGS. 11A-11C show that blockage of endogenous PGE2 signaling in muscleat an early time point of regeneration reduces regeneration and force.Endogenous MuSCs assayed in Pax7^(CreERT2);Rosa26-LSL-Luc mice treatedwith tamoxifen (TAM) by non-invasive bioluminescence imaging (BLI) afterinjection with vehicle (−) or NSAID (Indomethacin) post-cardiotoxininjury into the Tibialis anterior (TA); (n=3 mice per condition). FIG.11A: Experimental scheme. FIG. 11B: BLI; (n=3 mice per condition). FIG.11C: Muscle twitch forces at day 14 post-notexin injury (n=8 forvehicle-treated and 10 for NSAID-treated). ANOVA test for groupcomparison and significant difference for the endpoint by Fisher's test(FIG. 11B). Mann-Whitney test (FIG. 11C). *P<0.05, **P<0.001,***P<0.0005, and ****P<0.0001. Means+s.e.m.

FIGS. 12A-12K show that a transient increase in PGE2 in damaged muscletissues accelerates MuSC proliferation. FIG. 12A: Expression of Ptger4in freshly isolated muscle stem cells (MuSCs) from uninjured mousehindlimbs (Fresh MuSCs), MuSCs cultured for two days on hydrogels(Cultured MuSCs), primary myoblasts cultured in growth medium (MyoblastsGM) and differentiating primary myoblasts cultured in differentiationmedium for 24 hr (Myoblasts DM) (n=3 biological replicates percondition). FIG. 12B: PGE2 levels assayed by ELISA after tibialisanterior (TA) muscle injury with notexin; (n=4 mice per conditionmeasured). Control refers to the contralateral uninjured leg. FIG. 12C:Representative TA cross-sections of 3 and 6 days post-notexin injury.DAPI, blue; LAMININ, white; PGE2, green. Bar=40 μm. FIG. 12D: Expressionof prostaglandin synthetizing enzymes, Ptges and Ptges2 after TA muscleinjury (notexin) (n=3 mice with 2 technical replicates). Control refersto the contralateral uninjured leg. FIG. 12E: PGE2 levels of conditionedmedium from isolated fibers in the presence or absence of indomethacin(Indo) assayed by ELISA; (n=3 mice per condition). FIG. 12F: MuSCnumbers after 24 hr treatment with vehicle or PGE2 (10 ng/ml), andsubsequent culture on hydrogel until day 7; (n=12 mice in 4 independentexperiments). FIG. 12G: Trajectories of a MuSC clone treated withvehicle (top) or PGE2 (bottom) by time-lapse microscopy for 38 hr. FIG.12H: Change in MuSC cell counts (numbers) in clones tracked bytime-lapse microscopy after vehicle (left, n=40 clones) and PGE2treatment (right, n=44 clones). FIG. 12I: Plot of time to division afterplating for each MuSC clone treated with vehicle or PGE2. Clones showinga 38 hr time to division refers to clones that never divided during therecorded time-lapse. The lines represent the non-linear regression curvefrom Gaussian lognormal fit with R²=0.9 (control) and 0.97 (PGE2).FIG.12J: Violin plot of time to division post-plating in MuSC clones treatedwith vehicle or PGE2. FIG. 12K: Cell sizes of tracked MuSCs treated withvehicle or PGE2. *P<0.05, **P<0.001, ***P<0.0005 ****P<0.0001.Mann-Whitney test (FIGS. 12A, 12E, 12J, 12K); ANOVA test with Bonferronicorrection for multiple comparisons (FIGS. 12B, 12D); Paired t-test(FIG. 12F). Means+s.e.m.

FIGS. 13A-13G show that PGE2 treatment augments muscle regeneration.FIG. 13A: Engraftment of freshly sorted GFP/luc-labeled MuSCs (250cells) coinjected with vehicle or PGE2. Transplant scheme (top).Bioluminescence imaging (BLI) signals post-transplant expressed asaverage radiance (p s⁻¹ cm⁻² sr⁻¹); (n=4 and n=5 mice for vehicle andPGE2 treated respectively, bottom). At 4 weeks post-transplant,recipient mice were reinjured with Notexin. FIGS. 13B-13E: TAs of micewere injected with vehicle or PGE2 post-cardiotoxin (CTX) injury; (n=3mice per condition, vehicle-treated is the contralateral leg). FIG. 13B:Experimental scheme (top). Representative TA cross-section (bottom).DAPI, blue; LAMININ, green; PAX7, red. Arrowheads indicate PAX7⁺ MuSCs.Bar=40 μm. FIG. 13C: Quantification of PAX7⁺ satellite cells per 100fibers. FIG. 13D: Representative myofiber cross-sectional areas (CSA) invehicle (open white bar) and PGE2 treated (filled blue bar) TAs. FIG.13E Distribution of small (<1,000 μm² CSA) and large (>1,000 μm² CSA)myofibers. FIGS. 13F and 13G: Endogenous MuSCs assayed inPax7^(CreERT2);Rosa26-LSL-Luc mice treated with tamoxifen (TAM) by BLI;(n=3 mice per condition). FIG. 13F: Experimental scheme. FIG. 13G BLI(left); (n=3 mice per condition). Representative BLI image (right).Bar=5 mm. *P<0.05, **P<0.001. ANOVA test for group comparisons andsignificant difference for endpoint by Fisher's test (FIGS. 13A, 13G);Paired t-test (FIGS. 13C, 13E). Means+s.e.m.

FIGS. 14A-14E show that EP4 mediates PGE2 signaling in MuSCs. FIG. 14A:Expression of prostaglandin receptors (Ptger 1-4) by MuSCs after 24 hrtreatment with vehicle or PGE2; (n=3 mice with 2 technical replicates).FIG. 14B: cAMP levels in MuSCs after 1 hr PGE2 treatment; (n=6 mice with3 technical replicates assayed in 2 independent experiments). FIG. 14C:MuSC numbers after 24 hr treatment with vehicle or PGE2 in the absenceor presence of EP4 antagonist (ONO-AE3-208, 1 μM). FIG. 14D:Proliferation of EP4 null MuSCs treated with vehicle or PGE2. EP4^(f/f)MuSCs were treated with lentiviral vector encoding Cre (+Cre, EP4-null)or without (−Cre; control) to delete EP4 alelles. Scheme depictingEP4-null and control MuSC analysis (top). EP4-null and control MuSCnumbers; (n=6 mice in 2 independent experiments) (bottom). FIG. 14E:Engraftment of GFP/luc-labeled EP4^(f/f) MuSCs (1,000 cells) treatedwith Cre (+Cre) or without (−Cre; empty vector) in culture to delete EP4alelles. EP4^(f/f) MuSCs were transduced with a lentiviral vectorencoding GFP/luciferase for BLI. Transplant scheme (top). BLI signalspost transplant (n=5 mice per condition) (bottom left). RepresentativeBLI image (bottom right). Bar=5 mm. *P<0.05, **P<0.001, ****P<0.0001.Mann-Whitney test (FIGS. 14A, 14B); ANOVA test with Bonferronicorrection for multiple comparisons (FIGS. 14C, 14D); ANOVA test forgroup comparisons and significant difference for endpoint by Fisher'stest (FIG. 14E). Means+s.e.m. n.s., non significant.

FIGS. 15A-15G show that Nurr1 is a downstream effector of PGE2/EP4signaling in MuSCs. FIG. 15A: Heat map of differentially expressedtranscription factors in vehicle or PGE2 treated MuSCs after 24 hr. FIG.15B: Expression of Nurr1 after TA muscle injury (notexin) (n=3 mice pertimepoint). FIG. 15C: Expression of Nurr1 by MuSCs after 24 hr treatmentwith vehicle or PGE2; (n=3 mice, performed in 3 independentexperiments). FIG. 15D: Flow cytometric analysis of NURR1 or IgG controlin myogenic progenitors treated with vehicle or PGE2 for 24 hr. FIG.15E: MuSC numbers after 24 hr treatment of PGE2 or vehicle andsubsequent culture on hydrogel until day 7 of shSCR or shNurr1transfected cells (n=6 mice performed 2 independent experiments). FIG.15F: Expression of Nurr1 in Pax7^(CreERT2);EP4^(f/f) (EP4 cKO) MuSCstreated with or without 4-hydroxytamoxifen (4OHT) in vitro andsubsequently exposed to vehicle or PGE2 for 24 hr; (n=3 mice). FIG. 15G:Expression of Nurr1 in MuSCs, primary myoblasts cultured in growthmedium (Myob. GM) and differentiating primary myoblasts cultured indifferentiation medium for 24 hr (Myob. DM) (n=3 biological replicatesper condition). *P<0.05, **P<0.001, ***P<0.0005. ANOVA test withBonferroni correction for multiple comparisons (FIGS. 15B, 15E, 15F,15G); Mann-Whitney test (FIG. 15C). Means+s.e.m. n.s., non significant.

FIGS. 16A-16L show that loss of function of PGE2 signaling in MuSCsimpairs muscle regeneration and strength. FIGS. 16A-16H: Tibialisanteriors (TAs) of Pax7-specific EP4 conditional knockout mice(Pax7^(CreERT2);EP4^(f/f), EP4 cKO) treated with tamoxifen (TAM) wereassayed at 7 (FIGS. 16C, 16E), 14 (FIGS. 16G, 16H) and 21 (FIGS. 16B,16D) days post-notexin injury; (n=3 mice per condition for alltimepoints). FIG. 16A: Experimental scheme. FIG. 16B: Expression ofPtger4 (EP4 receptor) in sorted MuSCs (α⁷⁺ CD34⁺ lin⁻) from control orEP4 cKO mice 21 days post-injury. FIG. 16C: Percentage of embryonicMyosin Heavy Chain (eMyHC) positive fibers 7 days post-injury. FIG. 16D:Myofiber cross-sectional areas (CSA) in control and EP4 cKO TAs 21 dayspost-injury. FIG. 16E: Representative TA cross-section at 7 dayspost-injury, DAPI, blue; eMyHC, red (left); and at 21 days post-injury,DAPI, blue, LAMININ, green (right). Bar=40 μm. FIG. 16F: In vivo musclecontractile force assay scheme. FIG. 16G: Representative twitch force(left) and tetanic force (right) at day 14 post-notexin injury. FIG.16H: Quantification of muscle twitch forces (left) and tetanic forces(right). (n=8 for control and 3 for EP4 cKO). (FIGS. 16I, 16J)Endogenous muscle stem cells (MuSCs) assayed inPax7^(CreERT2);Rosa26-LSL-Luc mice treated with tamoxifen (TAM) bynon-invasive bioluminescence imaging (BLI) after injection with vehicleor NSAID (Indomethacin) post-cardiotoxin injury into the TA. FIG. 16I:Experimental scheme (top). Representative BLI image (bottom). Bar=5 mm.FIG. 16J: BLI; (n=3 mice per condition performed in 2 independentexperiments; figure is representative of one experiment). (FIGS. 16K,16L) Muscle force was measured after vehicle or NSAID (Indomethacin) atday 14 post-cardiotoxin in C57Bl/6 mice (2-4 month old). FIG. 16K:Representative twitch force. FIG. 16L: Quantification of muscle twitchforces (n=8 for vehicle-treated and 10 for NSAID-treated). *P<0.05,***P<0.0005 and ****P<0.0001. Mann-Whitney test (FIGS. 16B, 16C, 16H,16L); ANOVA test for group comparison and significant difference foreach bin by Fisher's test FIG. 16D, ANOVA test for group comparisons andsignificant difference for endpoint by Fisher's test FIG. 16J.Means+s.e.m.

FIG. 17 shows a model for PGE2 signaling to expand MuSC function inregeneration. Shown is achematic of the role of PGE2 in MuSCs. Afterinjury, PGE2 released into the muscle niche acts on the EP4 receptor,which signals through cAMP/phospho-CREB leading to the expression ofNurr1 proliferation-inducing transcription factor. This promotes MuSCexpansion for efficient muscle regeneration. Loss of PGE2/EP4 signalingby NSAID treatment or specific loss of EP4 receptor leads to aberrantMuSC function and impaired muscle regeneration and strength recovery.

FIGS. 18A-18G show that PGE2 promotes MuSC proliferation. FIG. 18A: PGE2levels assayed by ELISA after cryoinjury for tibialis anterior (TA);(n=3 mice per condition). Control refers to the contralateral uninjuredleg. FIG. 18B: Proliferation measured by the metabolic viability assayVisionBlue after treatment with vehicle or indicated doses of PGE2(1-200 ng/ml); (n=6 mice with 3 technical replicates in two independentexperiments). FIG. 18C: Representative image of MuSCs labeled with EdUduring 1 hr (red) and costained with MYOGENIN (green) after treatmentwith PGE2 (10 ng/ml) for 24 h or vehicle. Bar represents 40 μm. FIG.18D: Percentage of dividing MuSCs labeled with EdU in FIG. 18C; (n=6mice with 3 technical replicates in two independent experiments). FIG.18E: MuSC numbers after culture in growth medium with normal serum(non-str. Serum) or charcoal stripped medium (stripped-serum) treateddaily with vehicle or PGE2 for 7 days; (n=6 mice in 3 independentexperiments). FIG. 18F: Time to first division after plating for eachindividual MuSC clone analyzed by time-lapse after vehicle (left) orPGE2 (right) treatment. FIG. 18G: Cumulative Frequency of the time todivision after plating of all tracked MuSC clones throughout the entiretimelapse duration (38 hr).*P<0.05, **P<0.001. Mann-Whitney test (FIGS.18A, 18D). ANOVA test with Bonferroni correction for multiplecomparisons (FIG. 18E). Means+s.e.m. n.s., non significant.

FIGS. 19A-19E show that PGE2 direct injection augments muscleregeneration without promoting hypertrophy. FIG. 19A: Representativecross-sectional images of the TA showing a GFP⁺ MuSC after engraftmentof freshly sorted GFP/luc-labeled MuSCs (250 cells) coinjected with PGE2at 8 weeks post-engraftment. Images show staining with GFP, green,LAMININ, red and DAPI, blue. Bar represents 40 μm. FIG. 19B:Representative cross-sectional images of the TA showing GFP⁺ myofibersafter engraftment of freshly sorted GFP/luc-labeled MuSCs (250 cells)coinjected with PGE2 at 8 weeks post-engraftment. Images show stainingwith GFP, green, wheat germ agglutinin (WGA) or LAMININ, red and DAPI,blue. Bar represents 40 μm. FIG. 19C: Representative cross-sectionalimages of TA myofibers of C57Bl/6 mice at day 14 post-cardiotoxin (CTX)injury treated in vivo with vehicle or PGE2 48 hr post-injury. Imagesshow staining with LAMININ, green and DAPI, blue. Bar represents 40 μm.FIG. 19D: The corresponding segmentation images from (A) analyzed by theBaxter Algorithms for Myofiber Analysis to determine the cross sectionalarea (CSA) of transverse sections of myofibers (bottom) at day 14post-injury. Bar represents 40 μm. FIG. 19E: Mass of vehicle orPGE2-treated TAs at day 14 post-injury. Mann-Whitney test (FIG. 19E).Means+s.e.m. n.s., non-significant.

FIGS. 20A-20G show that EP4 loss of function in MuSC leads to decreasedproliferation. FIGS. 20A-20D: EP4^(f/f) MuSCs treated with lentiviralvector encoding mCherry/Cre (+Cre) or without (−Cre; empty vector) todelete EP4 alelles. Bar graphs show percentage of Cre⁺ MuSCs (FIG. 20A)and GFP/Luc⁺ MuSCs (FIG. 20B). FIG. 20C: Representative image. Bar=40μm; GFP, green; mCherry, red. FIG. 20D: Expression of Ptger4 byEP4^(f/f) MuSCs±Cre. FIGS. 20E-20G: Pax7 specific EP4 knockout MuSCsisolated from Pax7^(CreERT2);EP4^(f/f) or control Pax7^(+/+);EP4^(f/+)mice treated with 4-hydroxytamoxifen (4OHT) in vitro (n=3 mice percondition). FIG. 20E: Experimental scheme. FIG. 20F: MuSC numbers after7 days of culture. FIG. 20G: Expression of prostaglandin receptors(Ptger 1-4) by qRT-PCR. *P<0.05, **P<0.001, ***P<0.0005, ****P<0.0001.Mann-Whitney test (FIG. 20D); ANOVA test with Bonferroni correction formultiple comparisons (FIGS. 20F, 20G). Means+s.e.m. n.s.,non-significant.

FIGS. 21A-21E show transcriptome analysis of PGE2-treated MuSCs. FIG.21A: Heat map of the transcriptome of vehicle or PGE2 treated MuSCsafter 24 hr shown as expression fold-change over vehicle-treated MuSCs.FIG. 21B: Enriched molecular and cellular function pathways of thedifferentially expressed upregulated genes in the PGE2-treated MuSCsindicated by Ingenuity Pathway Analysis. FIG. 21C: Enriched pathway mapsof the differentially expressed upregulated genes in the PGE2-treatedMuSCs indicated by Metacore Analysis. FIG. 21D: Flow cytometric analysisof NURR1 in shSCR (control) or shNurr1 transfected cells. FIG. 21E:Expression of Ptger4 (EP4 receptor) in Pax7^(CreERT2):EP4^(f/f) MuSCstreated with or without 4-hydroxytamoxifen (4OHT) in vitro and andsubsequently exposed to vehicle or PGE2 for 24 hr; (n=3 mice).****P<0.0001. ANOVA test with Bonferroni correction for multiplecomparisons (FIG. 21E). Means+s.e.m. n.s., non-significant.

FIGS. 22A and 22B show that muscle mass is not altered after PGE2 lossof function post-injury. FIG. 22A: Mass of TAs of control orMuSC-specific EP4 conditional knockout mice at day 14 post-injury. FIG.22B: Mass of vehicle or NSAID-treated TAs at day 14 post-injury.Mann-Whitney test. Means+s.e.m. n.s., non significant.

FIGS. 23A-23C show that a composition comprising a combination of a PGE2derivative (16,16-dimethyl prostaglandin E2; dmPGE2) and bupivacaine(BPV) enhances muscle stem cell expansion during regeneration. FIG. 23Ashows a scheme illustrating experimental procedures for the in vivoanalysis of endogenous muscle stem cell (MuSC) expansion duringregeneration in Pax7^(CreERT2);Rosa26-LSL-Luc mice treated withtamoxifen (TAM) via bioluminescent imaging (BLI). FIG. 23B showsrepresentative BLI images of control (BPV/vehicle) and experimental(BPV/dmPGE2) mouse limb 2-week post-injury. Bar=5 mm. FIG. 23C showslog-fold changes of BLI signals between control and experimental groupsat week 2 post-injury. Data are shown as the mean±s.e.m. (n=6). Theasterisk (*) indicates statistical significant difference with p<0.05.

FIG. 24 shows a dose-dependent effect of bupivacaine, when administeredin combination with dmPGE2, in inducing muscle stem cell expansionduring regeneration. The graph shows the relative endogenous mousemuscle stem cell expansion in Pax7^(CreERT2);Rosa26-LSL-Luc mice asmeasured by the radiance fold change of bioluminescent imaging (BLI)from day 3 post-injection. Statistical significance for the differencebetween the control vs. treatment group was determined by one-way ANOVAtest with Bonferroni's multiple comparison correction. Error barrepresents s.e.m. and n>3 per condition.

FIGS. 25A-25D describe a handheld microendoscope for use in assessingthe benefits of compositions and methods of the present invention. FIG.25A shows photographs of the microendoscope and associated workstation.FIG. 25B shows a schematic of the microendoscope. FIG. 25C shows anexemplary image generated by the microendoscope. FIG. 25D shows a morehighly magnified exemplary image generated by the microendoscope.

FIG. 26 shows an exemplary timeline for a clinical trial.

FIGS. 27A-27C show the synergistic effect of combining a PGE2 compoundand a myotoxin to induce muscle regeneration and improve musclefunction. Pax7-CreERT2; Rosa-LSL-Luciferase mice (2-4 months old) weretreated with tamoxifen for five consecutive days in order to obtain Pax7promoter expressing luciferase mice in vivo. One week later, baselinetetanic force of the tibialis anterior was measured using a foot plateforce measurement instrument before injection of drugs (timepoint day0). Mice were subsequently injected with 50 μl of vehicle (saline), themuscle stem cell activator prostaglandin E2 (PGE2, 20 μg), the musclestem cell expansion agent bupivacaine (BPV, 0.25%) or the combinationdrug (bupivacaine 0.25% together with PGE2 20 μg) into the Tibialisanterior (TA) muscle. FIG. 27A shows bioluminescence (BLI, measured asradiance) measured every 3 days for 2 weeks to measure muscle stem cellexpansion. FIG. 27B shows the resulting tetanic force measured at week 4from the same mice, where the percent difference to baseline force wascalculated. FIG. 27C: at 4 weeks (endpoint) the TA was isolated, and thespecific force (mN/mm²) was obtained based on the physiologicalcross-sectional area (PCSA) calculated by the muscle length, weight andpennation angle. The specific force and the percent difference oftetanic force were significantly increased for the combination drugcompared to the vehicle and both of the small molecules injected alone.*P<0.05, **P<0.001. ANOVA test for group comparisons and significantdifference for endpoint by Fisher's test (FIG. 27A). ANOVA test withBonferroni correction for multiple comparisons (FIG. 27B, FIG. 27C).Data are shown as means±SEM.

DETAILED DESCRIPTION OF THE INVENTION I. INTRODUCTION

Recent studies have shown the importance of muscle stem cells (MuSCs) instimulating neuromuscular junctions in denervated muscles, althoughuntil recently improving the recovery of muscle function followingdenervation remained an unsolved problem. A solution to this problemlies in the ability to reverse or prevent denervation atrophy bystimulating and augmenting MuSCs that are already present in the musclesor by stimulating and augmenting MuSCs from muscle transplantation.

The present invention is based, in part, on the discovery that acombination of prostaglandin E2 (PGE2) compounds and myotoxins such asbupivacaine invoke dormant MuSCs to engage in muscle regeneration andrestore strength. In some cases, the addition of a myotoxin inducesmuscle regeneration. In those cases, the addition of a mytoxin to a PGE2compound improves muscle regeneration, better than muscle regenerationinduced by a PGE2 compound alone. As such, in certain aspects, thecompositions and methods of the present invention are particularlyuseful for promoting regeneration of atrophic abductor pollicis brevis(APB) muscle post nerve release surgery to promote neuromuscularjunction establishment and restoration of muscle contractile functionand volume.

Recent studies have shown the importance of muscle stem cells (MuSCs) instimulating neuromuscular junctions in denervated muscles (Liu et al.,2015), although until recently improving the recovery of muscle functionfollowing denervation remained an unsolved problem. A solution to thisproblem lies in the ability to reverse or prevent denervation atrophy bystimulating and augmenting MuSCs that are already present in themuscles.

II. DEFINITIONS

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forinstance, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the agent” includes reference to one or more agents knownto those skilled in the art, and so forth.

The term “prostaglandin E2” or “PGE2” refers to prostaglandin that canbe synthesized via arachidonic acid via cyclooxygenase (COX) enzymes andterminal prostaglandin E synthases (PGES). PGE2 plays a role in a numberof biological functions including vasodilation, inflammation, andmodulation of sleep/wake cycles.

The term “prostaglandin E2 receptor agonist” or “PGE2 receptor agonist”refers to a chemical compound, small molecule, polypeptide, biologicalproduct, etc. that can bind to and activate any PGE2 receptor, therebystimulating the PGE2 signaling pathway.

The term “compound that attenuates PGE2 catabolism” refers to a chemicalcompound, small molecule, polypeptide, biological product, etc. that canreduce or decrease the breakdown of PGE2.

The term “compound that neutralizes PGE2 inhibition” refers to achemical compound, small molecule, polypeptide, biological product, etc.that can block or impede an inhibitor of PGE2 synthesis, activity,secretion, function, and the like.

The term “compound that attenuates PGE2 catabolism” refers to a physicalprocess that attenuates the transport of PGE2 via a transporter for thebreakdown of PGE2 intracellularly. This process can be the physicalblock of a prostaglandin transporter, which transports PGE2 inside cellsfor catabolism by 15-PGDH. The prostaglandin transporter is also knownas 2310021C19Rik, MATR1, Matrin F/Q, OATP2A1, PGT, PHOAR2, SLC21A2,solute carrier organic anion transporter family member 2A1, and SLCO2A1.

The term “derivative,” in the context of a compound, includes but is notlimited to, amide, ether, ester, amino, carboxyl, acetyl, and/or alcoholderivatives of a given compound.

The term “treating” or “treatment” refers to any one of the following:ameliorating one or more symptoms of disease; preventing themanifestation of such symptoms before they occur; slowing down orcompletely preventing the progression of the disease (as may be evidentby longer periods between reoccurrence episodes, slowing down orprevention of the deterioration of symptoms, etc.); enhancing the onsetof a remission period; slowing down the irreversible damage caused inthe progressive-chronic stage of the disease (both in the primary andsecondary stages); delaying the onset of said progressive stage; or anycombination thereof.

The term “administer,” “administering,” or “administration” refers tothe methods that may be used to enable delivery of agents orcompositions such as the compounds and cells described herein to adesired site of biological action. These methods include, but are notlimited to, parenteral administration (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular, intra-arterial, intravascular,intracardiac, intrathecal, intranasal, intradermal, intravitreal, andthe like), transmucosal injection, oral administration, administrationas a suppository, and topical administration. One skilled in the artwill know of additional methods for administering a therapeuticallyeffective amount of the compounds and/or cells described herein forpreventing or relieving one or more symptoms associated with a diseaseor condition.

The term “therapeutically effective amount” or “therapeuticallyeffective dose” or “effective amount” refers to an amount of a compound,therapeutic agent (e.g., cells), and/or pharmaceutical drug that issufficient to bring about a beneficial or desired clinical effect. Atherapeutically effective amount or dose may be based on factorsindividual to each patient, including, but not limited to, the patient'sage, size, type or extent of disease, stage of the disease, route ofadministration of the regenerative cells, the type or extent ofsupplemental therapy used, ongoing disease process and type of treatmentdesired (e.g., aggressive vs. conventional treatment). Therapeuticallyeffective amounts of a pharmaceutical compound or compositions, asdescribed herein, can be estimated initially from cell culture andanimal models. For example, IC₅₀ values determined in cell culturemethods can serve as a starting point in animal models, while IC₅₀values determined in animal models can be used to find a therapeuticallyeffective dose in humans.

The term “pharmaceutically acceptable carrier” refers to a carrier or adiluent that does not cause significant irritation to an organism anddoes not abrogate the biological activity and properties of theadministered compound.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, rats, simians, humans, farm animals, sport animals, and pets.

The term “mechanical injury” refers to myotoxic effects that are inducedby physical processes, non-limiting examples including cutting, burning,freezing, needle puncture, and exercise. In some instances, mechanicalinjury occurs as the result of a surgical procedure (e.g., a surgicalprocedure or treatment that comprises cutting, incising, suturing,and/or repairing a muscle) or a traumatic injury (e.g., accidentaltrauma or injury), non-limiting examples being blunt and/or crushinjuries (e.g., involving limbs or appendages such as the arms, legs,hand, feet, and digits).

The term “myotoxin” means a compound that induces damage or death inmuscle cells. In some embodiments, the toxic effects produced in musclecells (e.g., muscle cell damage, muscle cell death) by the myotoxin maytrigger, either directly or indirectly, the subsequent activation ofmuscle stem cells, muscle regeneration, or both. Non-limiting examplesof myotoxins include anesthetics (e.g., bupivacaine), divalent cations,snake venom, lizard venom, bee venom, and compounds contained within(e.g., notexin, cardiotoxin, and bungarotoxin).

In some cases, the myotoxin is a mild myotoxin. A mild myotoxin as usedherein means that muscle tissue is subjected to minor damage assessed byhistology. A mild myotoxin as used herein may include any compound thatcauses transient but reversible muscle damage or cell death. Myotoxicityof excipients administered intramuscularly can be assessed by monitoringrecruitment of inflammatory cells (leucocytes, macrophages and othermonocytes), induction of transient cytokine levels, growth factors andinflammatory metabolites. Histologically, myotoxicity could be assessedby disruption of myofiner architecture and the encompassing matrix,induction of acute cell death and necrosis, induction of acute muscleresident cell proliferation (including muscle stem cells), induction ofembryonic myosin heavy chain (eMHC) expression, and presence of centralnuclei position within myofibers. Systemically, myotoxicity can also bedetected by muscle creatin kinase level. Functionally, myotoxicity canbe detected by reduction in muscle force and disruption in meuromuscularjunction.

Reversibility of the myotoxicity can be assessed by restoration in thedamaged myofiber histology in a short duration (for example, in about 21days in a mouse model), lack of fibrosis in the tissue (lack of excesscollagen deposition or other matrix constituents) and lack of lipiddeposition (adipocyte transdifferentiation).

Non-limiting examples of mild myotoxins may include anesthetics, suchas, e.g., bupivacaine or lidocaine.

The term “acute exposure,” in the context of administration of acompound, refers to a temporary or brief application of a compound to asubject, e.g., human subject, or cells. In some embodiments, an acuteexposure includes a single administration of a compound over the courseof treatment or over an extended period of time.

The term “intermittent exposure,” in the context of administration of acompound, refers to a repeated application of a compound to a subject,e.g., human subject, or cells, wherein a desired period of time lapsesbetween applications.

The term “acute regimen,” in the context of administration of acompound, refers to a temporary or brief application of a compound to asubject, e.g., human subject, or to a repeated application of a compoundto a subject, e.g., human subject, wherein a desired period of time(e.g., 1 day) lapses between applications. In some embodiments, an acuteregimen includes an acute exposure (e.g., a single dose) of a compoundto a subject over the course of treatment or over an extended period oftime. In other embodiments, an acute regimen includes intermittentexposure (e.g., repeated doses) of a compound to a subject in which adesired period of time lapses between each exposure.

The term “continuous exposure,” in the context of administration of acompound, refers to a repeated, chronic application of a compound to asubject, e.g., human subject, or cells, over an extended period of time.

The term “chronic regimen,” in the context of administration of acompound, refers to a repeated, chronic application of a compound to asubject, e.g., human subject, over an extended period of time such thatthe amount or level of the compound is substantially constant over aselected time period. In some embodiments, a chronic regimen includes acontinuous exposure of a compound to a subject over an extended periodof time.

III. DETAILED DESCRIPTION OF THE EMBODIMENTS

A. Compositions and Pharmaceutical Compositions

In one aspect of the present invention, provided herein is a compositionfor preventing or treating a muscle condition. In some embodiments, thecomposition comprises a prostaglandin E2 (PGE2) compound and a myotoxin.In some embodiments, the PGE2 compound is selected from the groupconsisting of PGE2, a PGE2 prodrug, a PGE2 receptor agonist, a compoundthat attenuates PGE2 catabolism, a compound that neutralizes PGE2inhibition, a derivative thereof, an analog thereof, and a combinationthereof. A prodrug of PGE2 can be metabolized into a pharmacologicallyactive PGE2 drug, for example, at the site of administration or muscleregeneration, or when the prodrug is exposed to muscle cells.

In particular embodiments, the PGE2 compound is a PGE2 derivative oranalog that contains one or more modifications to PGE2 that increase itsstability, activity, resistance to degradation, transport into musclecells (e.g., promote cellular uptake), and/or retention in muscle cells(e.g., reduce secretion from muscle cells after uptake).

Without limitation, examples of PGE2 derivatives and analogs include2,2-difluoro-16-phenoxy-PGE2 compounds,2-decarboxy-2-hydroxymethyl-16-fluoro-PGE2 compounds,2-decarboxy-2-hydroxymethyl-11-deoxy-PGE2 compounds, 19(R)-hydroxy PGE2,16,16-dimethyl PGE2, 16,16-dimethyl PGE2 p-(p-acetamidobenzamido) phenylester, 11-deoxy-16,16-dimethyl PGE2 (dmPGE2),9-deoxy-9-methylene-16,16-dimethyl PGE2, 9-deoxy-9-methylene PGE2,butaprost, sulprostone, enprostil, PGE2 serinol amide, PGE2 methylester, 16-phenyl tetranor PGE2, 5-trans-PGE2, 15(S)-15-methyl PGE2, and15(R)-15-methyl PGE2. Additional PGE2 derivatives and analogs are setforth, e.g., in U.S. Pat. No. 5,409,911.

Additional non-limiting examples of PGE2 derivatives and analogs includehydantoin derivatives of PGE2, the more stable PGE2 analogs described inZhao et al. (Bioorganic & Medicinal Chemistry Letters, 17:6572-5 (2007))in which the hydroxy cyclopentanone ring is replaced by heterocyclicrings and the unsaturated alpha-alkenyl chain is substituted with aphenethyl chain, the PGE2 analogs described in Ungrin et al. (Mol.Pharmacol., 59:1446-56 (2001)), the 13-dehydro derivatives of PGE2described in Tanami et al. (Bioorg. Med. Chem. Lett., 8:1507-10 (1998)),and the substituted cyclopentanes described in U.S. Pat. Nos. 8,546,603and 8,158,676.

In some embodiments, the PGE2 compound is an agonist of a PGE2 receptor,e.g., EP1 receptor, EP2 receptor, EP3 receptor, and EP4 receptor.Non-limiting examples of PGE2 receptor agonists include ONO-DI-004,ONO-AE1-259, ONO-AE-248, ONO-AE1-329, ONO-4819CD (Ono PharmaceuticalCo., Japan), L-902688 (Cayman Chemical), CAY10598 (Cayman Chemical), andCP-533536 (Pfizer). Additional PGE2 receptor agonists are described,e.g., in U.S. Pat. Nos. 6,410,591; 6,610,719; 6,747,037; 7,696,235;7,662,839; 7,652,063; 7622,475; and 7,608,637.

In particular embodiments, the PGE2 receptor agonist comprises acompound of Formula (I), a derivative thereof, a pharmaceuticallyacceptable salt thereof, a solvate thereof, a stereoisomer thereof, or acombination thereof,

wherein ring A is a substituted 4- to 6-membered cycloalkyl ring or asubstituted 4- to 6-membered cycloalkenyl ring that comprisessubstituents R¹ and R² that are independently selected from the groupconsisting of substituted C₁-C₁₀ alkyl and substituted C₂-C₁₀ alkenyl,and ring A further comprises one or more additional substituents. Insome embodiments, ring A is a substituted cyclopentyl ring or asubstituted cyclopentenyl ring. In particular embodiments, the one ormore additional substituents on ring A are selected from the groupconsisting of deuterium, hydroxy, amino, oxo, C₁-C₆ alkyl, and halogen.In some instances, the one or more additional substituents on ring A arehydroxy or oxo. In some embodiments, ring A has two additionalsubstituents that are taken together to form a covalent bond to form aheterocycloalkyl ring.

In some embodiments, ring A is selected from the consisting of

In particular embodiments, ring A is selected from the group consistingof from the group consisting of

In some instances, ring A is

In some embodiments, R¹ is substituted C₁-C₁₀ alkyl. In otherembodiments, R¹ is substituted C₂-C₁₀ alkenyl. In some embodiments, R¹is selected from the group consisting of deuterium, hydroxy, oxo, C₁-C₆alkyl, —COOR³, and halogen, wherein R³ is hydrogen or C₁-C₆ alkyl.

In some embodiments, R¹ is selected from the group consisting of

In other embodiments, R¹ is selected from the group consisting of

In some instances, R¹ is

In some embodiments, R² is substituted C₁-C₁₀ alkyl. In otherembodiments, R² is substituted C₂-C₁₀ alkenyl. In some embodiments, thesubstituent on R² is selected from the group consisting of deuterium,hydroxy, oxo, C₁-C₆ alkyl, —COOR³, and halogen, wherein R³ is hydrogenor C₁-C₆ alkyl.

In some embodiments, R² is selected from the group consisting of

In some embodiments, R² is selected from the group consisting of

In some instances, R² is

In some embodiments, the compound of Formula (I), the pharmaceuticallyacceptable salt thereof, the solvate thereof, or the stereoisomerthereof is a compound of Formula (Ia), Formula (Ib), Formula (Ic), orFormula (Id), or is a pharmaceutically acceptable salt thereof, asolvate thereof, or a stereoisomer thereof:

In some instances, the compound is of Formula (Id).

In some embodiments, the PGE2 compound is PGE2. In other embodiments,the PGE2 compound is a derivative of PGE2. In some instances, thederivative is 16,16-dimethyl prostaglandin E2 (dmPGE2). In particularembodiments, the PGE2 compound is PGE2 and/or dmPGE2.

In other embodiments, the PGE2 compound is a derivative of PGE2. In someinstances, the derivative is PGE2 conjugated to a moiety. In particularembodiments, the PGE2 compound is PGE2-Biotin or PGE2-PEG (Polyethyleneglycol) hydrogel. Exemplary embodiments are shown below:

In some embodiments, the PGE2 derivative comprises a PGE2 compoundconjugated to a molecular probe. In some cases, the molecular probe is apeptide sequence, a fragment antigen-binding (Fab), a heavy-chain onlyantibody (HcAbs), a full-length antibody (Ab), a single-domainantibody/nanobody (Nb), or a nanoparticle, or a combination. In somecases, the molecular probe is capable of homing to and targeting muscletissue via systemic delivery.

In those embodiments, a PGE2 derivative comprising a PGE2 compoundconjugated to a molecular probe may increase the half-life of the PGE2compound, increase the specificity of the PGE compound, and reduceadverse off-target effects of the PGE2 compund. Non-limiting examples ofPGE2 conjugated to a molecular probe include PGE2-Integrin-alpha7antibody or nanobody; PGE2-M-cadherin antibody or nanobody; andPGE2-anti PGE2 antibody. In some cases, a PGE2 derivative comprising aPGE2 compound conjugated to a molecular probe may be used to treatsarcopenia or cachexia.

In some embodiments, the PGE2 compound is a compound that attenuatesPGE2 catabolism. In some cases, a compound that attenuates PGE2catabolism can be a compound, a neutralizing peptide, or a neutralizingantibody that inactivates or blocks 15-hydroxyprostaglandindehydrogenase (15-PGDH) or inactivates or blocks a prostaglandintransporter, which transports PGE2 inside cells for catabolism by15-PGDH. The prostaglandin transporter is also known as 2310021C19Rik,MATR1, Matrin F/Q, OATP2A1, PGT, PHOAR2, SLC21A2, solute carrier organicanion transporter family member 2A1, and SLCO2A1.

In some embodiments, the composition may include a stem-cell inducingmolecule. In some cases, the stem-cell inducing molecule is a PGE2compound as described herein. In some cases, the composition includes astem-cell inducing molecule in combination with a myotoxin. Othernon-limiting examples of stem-cell inducing molecules that may be usedherein include oxytocin, beta integrin activating antibody, rapamycin,SetD7 inhibitors, p38 MAPK inhibitors (such as SB202190 and SB203580),neuregulin, nerve growth factor (NGF), Hif2alpha inhibitors, basicfibroblast growth factor (bFGF), fibroblast growth factor 4 (FGF4),epidermal growth factor (EGF), Interleukin-1α, Interleukin-13, TNFα,LIF, IL6, interferon gamma, oncostatin M (OSM), ghrelin, and apelin.

In some embodiments, the myotoxin is selected from the group consistingof an anesthetic, a divalent cation, venom from snakes, venom fromlizards, venom from bees, and a combination thereof. Suitable divalentcations include but are not limited to Ba²⁺, Sr²⁺, Mg²⁺, Ca²⁺, Mn²⁺,Ni²⁺, Co²⁺, salts thereof, and combinations thereof. In someembodiments, the snake or lizard venom is selected from the groupconsisting of notexin, cardiotoxin, bungarotoxin, and a combinationthereof.

In some embodiments, the anesthetic is selected from the groupconsisting of an amino-amide anesthetic, an amino-ester anesthetic, anda combination thereof. In some cases, the anesthetic is a mild myotoxin.Non-limiting examples of amino-amide anesthetics include bupivacaine,levobupivacaine, articaine, ropivacaine, butanilicaine, carticaine,dibucaine, etidocaine, lidocaine, mepivacaine, prilocaine, andtrimecaine. In some embodiments, the composition comprises a combinationof amino-amide anesthetics.

In some embodiments, the anesthetic is an amino-ester anesthetic. Inparticular embodiments, the amino-ester anesthetic is an aminobenzoicacid ester anesthetic, a benzoic acid ester anesthetic, or a combinationthereof. Non-limiting examples of aminobenzoic acid ester anestheticsinclude benzocaine, butacaine, butamben, chloroprocaine, dimethocaine,lucaine, meprylcaine, metabutethamine, metabutoxycaine, nitracaine,orthocaine, propoxycaine, procaine, proxymetacaine, risocaine, andtetracaine. Non-limiting examples of benzoic acid anesthetics includeamylocaine, cocaine, cyclomethycaine, α-eucaine, β-eucaine, hexylcaine,isobucaine, and piperocaine. In particular embodiments, the compositioncomprises a combination of one or more aminobenzoic acid esteranesthetics and/or one or more benzoic acid ester anesthetics.

Other non-limiting examples of anesthetics that may have mild myotoxiceffects include benzonatate, diperodon, fomocaine, fotocaine,hydroxyprocaine, oxetacaine, oxybuprocaine, paraethoxycaine, phenacaine,piridocaine, pramocaine, primacaine, procainamide, proparacaine,pyrrocaine, quinisocaine, tolycaine, and tropacocaine.

In some embodiments, the composition comprises a PGE2 compound thatcomprises PGE2 and/or dmPGE2 and a myotoxin that is bupivacaine.

Compositions of the present invention may be suitable for treating anynumber of muscle conditions, including but not limited to muscleconditions that are associated with muscle damage, injury, or atrophy.The compositions are also useful for promoting muscle regeneration in asubject in need thereof, for increasing muscle mass in a subject in needthereof, or both. Non-limiting examples of suitable conditions forprevention or treatment with compositions of the present inventioninclude traumatic injury (e.g., acute muscle trauma, acute nervetrauma), acute muscle injury, acute nerve injury, chronic nerve injury,soft tissue hand injury, carpal tunnel syndrome (CTS), Duchenne musculardystrophy (DMD), Becker muscular dystrophy, limb girdle musculardystrophy, amyotrophic lateral sclerosis (ALS), distal musculardystrophy (DD), inherited myopathies, myotonic muscular dystrophy (MDD),mitochondrial myopathies, myotubular myopathy (MM), myasthenia gravis(MG), congestive heart failure, periodic paralysis, polymyositis,rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiaccachexia, stress induced urinary incontinence, sarcopenia, spinalmuscular atrophy, fecal sphincter dysfunction, Bell's palsy, rotatorcuff injury, spinal cord injury, hip replacement, knee replacement,wrist fracture, diabetic neuropathy, gastroesophageal reflux disease(GERD), obstructive sleep apnea (OSA), pelvic floor disorders (e.g.,stress urinary incontinence, overactive bladder/urinary urgencyincontinence, mixed urinary incontinence, pelvic organ prolapse, fecalincontinence), musculoskeletal disorders (e.g., impaired hand function,impaired thumb function, impaired foot function), plantar fasciitis,foot drop, disuse-induced muscle atrophy, impaired eyelid function(e.g., eyelid drooping, impaired blinking, entropion, ectropion),strabismus, nystagmus, and presbyopia. Additional examples of suitableconditions for prevention or treatment with compositions of the presentinvention may include muscle disorders that affect small isolatedmuscles that can be regenerated with localized transplantation of smallnumbers of cells, including: atrophy and muscle dysfunction in the faceor hand after nerve injury or direct trauma that does not recover afterreinnervation; extraocular muscle injury causing inability to move theeye and dipoplia seen in Graves' disease, traumatic injury, andprogressive external ophthalmoplegia; and urinary and fecalincontinence.

In another aspect of the present invention, provided herein is apharmaceutical composition. In some embodiments, the pharmaceuticalcomposition comprises a pharmaceutically acceptable carrier and acomposition described herein that comprises a PGE2 compound and amyotoxin. In certain aspects, pharmaceutically acceptable carriers aredetermined in part by the particular composition being administered, aswell as by the particular method used to administer the composition.Accordingly, there is a wide variety of suitable formulations ofpharmaceutical compositions of the present invention (see, e.g.,REMINGTON'S PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co.,Easton, Pa. (1990)).

As used herein, “pharmaceutically acceptable carrier” comprises any ofstandard pharmaceutically accepted carriers known to those of ordinaryskill in the art in formulating pharmaceutical compositions. Thus, thecells or compounds, by themselves, such as being present aspharmaceutically acceptable salts, or as conjugates, may be prepared asformulations in pharmaceutically acceptable diluents; for example,saline, phosphate buffer saline (PBS), aqueous ethanol, or solutions ofglucose, mannitol, dextran, propylene glycol, oils (e.g., vegetableoils, animal oils, synthetic oils, etc.), microcrystalline cellulose,carboxymethyl cellulose, hydroxylpropyl methyl cellulose, magnesiumstearate, calcium phosphate, gelatin, polysorbate 80 or the like, or assolid formulations in appropriate excipients.

The pharmaceutical compositions will often further comprise one or morebuffers (e.g., neutral buffered saline or phosphate buffered saline),carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol,proteins, polypeptides or amino acids such as glycine, antioxidants(e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene,butylated hydroxyanisole, etc.), bacteriostats, chelating agents such asEDTA or glutathione, solutes that render the formulation isotonic,hypotonic or weakly hypertonic with the blood of a recipient, suspendingagents, thickening agents, preservatives, flavoring agents, sweeteningagents, and coloring compounds as appropriate.

The pharmaceutical compositions of the invention may be administered ina manner compatible with the dosage formulation, and in such amount aswill be therapeutically effective. The quantity to be administered maydepend on a variety of factors including, e.g., the age, body weight,physical activity, and diet of the individual, the condition or diseaseto be treated, and the stage or severity of the condition or disease. Incertain embodiments, the size of the dose may also be determined by theexistence, nature, and extent of any adverse side effects that accompanythe administration of a therapeutic agent(s) in a particular individual.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular patient may be varied and maydepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, hereditary characteristics, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and the hostundergoing therapy.

In certain embodiments, the dose of the compound may take the form ofsolid, semi-solid, lyophilized powder, or liquid dosage forms, such as,for example, tablets, pills, pellets, capsules, powders, solutions,suspensions, emulsions, suppositories, retention enemas, creams,ointments, lotions, gels, aerosols, foams, or the like, preferably inunit dosage forms suitable for simple administration of precise dosages.

As used herein, the term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosages for humans and other mammals,each unit containing a predetermined quantity of a therapeutic agentcalculated to produce the desired onset, tolerability, and/ortherapeutic effects, in association with a suitable pharmaceuticalexcipient (e.g., an ampoule). In addition, more concentrated dosageforms may be prepared, from which the more dilute unit dosage forms maythen be produced. The more concentrated dosage forms thus may containsubstantially more than, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more times the amount of the therapeutic compound.

Methods for preparing such dosage forms are known to those skilled inthe art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra). Thedosage forms typically include a conventional pharmaceutical carrier orexcipient and may additionally include other medicinal agents, carriers,adjuvants, diluents, tissue permeation enhancers, solubilizers, and thelike. Appropriate excipients can be tailored to the particular dosageform and route of administration by methods well known in the art (see,e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra).

Examples of suitable excipients include, but are not limited to,lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,saline, syrup, methylcellulose, ethylcellulose,hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols,e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc. The dosage formscan additionally include lubricating agents such as talc, magnesiumstearate, and mineral oil; wetting agents; emulsifying agents;suspending agents; preserving agents such as methyl-, ethyl-, andpropyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents suchas inorganic and organic acids and bases; sweetening agents; andflavoring agents. The dosage forms may also comprise biodegradablepolymer beads, dextran, hydrogels, and cyclodextrin inclusion complexes.

For oral administration, the therapeutically effective dose can be inthe form of tablets, capsules, emulsions, suspensions, solutions,syrups, sprays, lozenges, powders, and sustained-release formulations.Suitable excipients for oral administration include pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesiumcarbonate, and the like.

The therapeutically effective dose can also be provided in a lyophilizedform. Such dosage forms may include a buffer, e.g., bicarbonate, forreconstitution prior to administration, or the buffer may be included inthe lyophilized dosage form for reconstitution with, e.g., water. Thelyophilized dosage form may further comprise a suitable vasoconstrictor,e.g., epinephrine. The lyophilized dosage form can be provided in asyringe, optionally packaged in combination with the buffer forreconstitution, such that the reconstituted dosage form can beimmediately administered to an individual.

In some embodiments, a pharmaceutical composition of the presentinvention comprises a pharmaceutically acceptable carrier that comprisesan aqueous base. In other embodiments, the pharmaceutically acceptablecarrier comprises a low viscosity compound. In some instances, the lowviscosity compound comprises gelatin. In other instances, the lowviscosity compound comprises a hydrogel.

B. Methods for Promoting Muscle Regeneration and Preventing or TreatingMuscle Conditions

In another aspect of the present invention, provided herein is a methodfor promoting muscle regeneration in a subject in need thereof,increasing muscle mass in a subject in need thereof, or both. In someembodiments, the method comprises administering a combination of a PGE2compound and a myotoxin to the subject. In some embodiments, apharmaceutical composition comprising a pharmaceutically acceptablecarrier and combination of a PGE2 compound and a myotoxin isadministered to the subject. In some embodiments, a therapeuticallyeffective amount of the PGE2 compound is administered to the subject. Inother embodiments, a therapeutically effective amount of the myotoxin isadministered to the subject. In particular embodiments, atherapeutically effective amount of the PGE2 compound and the myotoxinare administered to the subject.

In yet another aspect of the present invention, provided herein is amethod for preventing or treating a muscle condition in a subject inneed thereof. In some embodiments, the method comprises administering acombination of a PGE2 compound and a myotoxin to the subject. In someembodiments, a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and combination of a PGE2 compound and a myotoxin isadministered to the subject. In some embodiments, a therapeuticallyeffective amount of the PGE2 compound is administered to the subject. Inother embodiments, a therapeutically effective amount of the myotoxin isadministered to the subject. In particular embodiments, atherapeutically effective amount of the PGE2 compound and the myotoxinare administered to the subject.

In still another aspect of the present invention, provided herein is amethod for preventing or treating a muscle condition in a subject inneed thereof. In some embodiments, the method comprises administering aPGE2 receptor agonist to the subject. In other embodiments, the methodfurther comprises administering a myotoxin to the subject. In someembodiments, a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier, a PGE2 receptor agonist, and optionally a myotoxinis administered to the subject. In some embodiments, a therapeuticallyeffective amount of the PGE2 receptor agonist is administered to thesubject. In other embodiments, a therapeutically effective amount of themyotoxin is administered to the subject. In particular embodiments, atherapeutically effective amount of the PGE2 receptor agonist and themyotoxin are administered to the subject.

In some embodiments, the methods comprise administering a PGE2 compoundthat is selected from the group consisting of PGE2, a PGE2 prodrug (e.g.PGE2 coupled to neural cadherin (NCAD) that targets NCAD receptor), aPGE2 receptor agonist, a compound that attenuates PGE2 catabolism, acompound that neutralizes PGE2 inhibition, a derivative thereof, ananalog thereof, and a combination thereof. A prodrug of PGE2 can bemetabolized into a pharmacologically active PGE2 drug, for example, atthe site of administration or muscle regeneration, or when the prodrugis exposed to muscle cells. In some cases, the PGE2 compound is abiotinylated drug or other modification that retains PGE2 receptorengagement and signaling but prevents internalization—leading toprolonged activity and overcoming the degradative pathway.

In particular embodiments, the PGE2 compound that is administered is aPGE2 derivative or analog that contains one or more modifications toPGE2 that increase its stability, activity, resistance to degradation,transport into muscle cells (e.g., promote cellular uptake), and/orretention in muscle cells (e.g., reduce secretion from muscle cellsafter uptake).

Without limitation, examples of PGE2 derivatives and analogs that aresuitable for administration according to methods of the presentinvention include 2,2-difluoro-16-phenoxy-PGE2 compounds,2-decarboxy-2-hydroxymethyl-16-fluoro-PGE2 compounds,2-decarboxy-2-hydroxymethyl-11-deoxy-PGE2 compounds, 19(R)-hydroxy PGE2,16,16-dimethyl PGE2, 16,16-dimethyl PGE2 p-(p-acetamidobenzamido) phenylester, 11-deoxy-16,16-dimethyl PGE2 (dmPGE2),9-deoxy-9-methylene-16,16-dimethyl PGE2, 9-deoxy-9-methylene PGE2,butaprost, sulprostone, enprostil, PGE2 serinol amide, PGE2 methylester, 16-phenyl tetranor PGE2, 5-trans-PGE2, 15(S)-15-methyl PGE2, and15(R)-15-methyl PGE2, and PGE2-Biotin or PGE2-PEG (Polyethylene glycol)hydrogel. Additional PGE2 derivatives and analogs are set forth, e.g.,in U.S. Pat. No. 5,409,911.

Additional non-limiting examples of PGE2 derivatives and analogs foradministration include hydantoin derivatives of PGE2, the more stablePGE2 analogs described in Zhao et al. (Bioorganic & Medicinal ChemistryLetters, 17:6572-5 (2007)) in which the hydroxy cyclopentanone ring isreplaced by heterocyclic rings and the unsaturated alpha-alkenyl chainis substituted with a phenethyl chain, the PGE2 analogs described inUngrin et al. (Mol. Pharmacol., 59:1446-56 (2001)), the 13-dehydroderivatives of PGE2 described in Tanami et al. (Bioorg. Med. Chem.Lett., 8:1507-10 (1998)), and the substituted cyclopentanes described inU.S. Pat. Nos. 8,546,603 and 8,158,676.

In some embodiments, a PGE2 compound that is an agonist of a PGE2receptor is administered, e.g., EP1 receptor, EP2 receptor, EP3receptor, and EP4 receptor. Non-limiting examples of PGE2 receptoragonists include ONO-DI-004, ONO-AE1-259, ONO-AE-248, ONO-AE1-329,ONO-4819CD (Ono Pharmaceutical Co., Japan), L-902688 (Cayman Chemical),CAY10598 (Cayman Chemical), and CP-533536 (Pfizer). Additional PGE2receptor agonists are described, e.g., in U.S. Pat. Nos. 6,410,591;6,610,719; 6,747,037; 7,696,235; 7,662,839; 7,652,063; 7622,475; and7,608,637.

In particular embodiments, the PGE2 receptor agonist that isadministered per methods of the present invention comprises a compoundof Formula (I), a derivative thereof, a pharmaceutically acceptable saltthereof, a solvate thereof, a stereoisomer thereof, or a combinationthereof,

wherein ring A is a substituted 4- to 6-membered cycloalkyl ring or asubstituted 4- to 6-membered cycloalkenyl ring that comprisessubstituents R¹ and R² that are independently selected from the groupconsisting of substituted C₁-C₁₀ alkyl and substituted C₂-C₁₀ alkenyl,and ring A further comprises one or more additional substituents. Insome embodiments, ring A is a substituted cyclopentyl ring or asubstituted cyclopentenyl ring. In particular embodiments, the one ormore additional substituents on ring A are selected from the groupconsisting of deuterium, hydroxy, amino, oxo, C₁-C₆ alkyl, and halogen.In some instances, the one or more additional substituents on ring A arehydroxy or oxo. In some embodiments, ring A has two additionalsubstituents that are taken together to form a covalent bond to form aheterocycloalkyl ring.

In some embodiments, ring A is selected from the consisting of

In particular embodiments, ring A is selected from the group consistingof from the group consisting of

In some instances, ring A is

In some embodiments, R¹ is substituted C₁-C₁₀ alkyl. In otherembodiments, R¹ is substituted C₂-C₁₀ alkenyl. In some embodiments, R¹is selected from the group consisting of deuterium, hydroxy, oxo, C₁-C₆alkyl, —COOR³, and halogen, wherein R³ is hydrogen or C₁-C₆ alkyl.

In some embodiments, R¹ is selected from the group consisting of

In other embodiments, R¹ is selected from the group consisting of

In some instances, R¹ is

In some embodiments, R² is substituted C₁-C₁₀ alkyl. In otherembodiments, R² is substituted C₂-C₁₀ alkenyl. In some embodiments, thesubstituent on R² is selected from the group consisting of deuterium,hydroxy, oxo, C₁-C₆ alkyl, —COOR³, and halogen, wherein R³ is hydrogenor C₁-C₆ alkyl.

In some embodiments, R² is selected from the group consisting of

In some embodiments, R² is selected from the group consisting of

In some instances, R² is

In some embodiments, the compound of Formula (I), the pharmaceuticallyacceptable salt thereof, the solvate thereof, or the stereoisomerthereof is a compound of Formula (Ia), Formula (Ib), Formula (Ic), orFormula (Id), or is a pharmaceutically acceptable salt thereof, asolvate thereof, or a stereoisomer thereof:

In some instances, the compound is of Formula (Id).

In some embodiments, the PGE2 compound that is administered according tothe methods of the present invention comprises PGE2. In otherembodiments, the PGE2 compound that is administered comprises aderivative of PGE2. In some instances, the derivative is 16,16-dimethylprostaglandin E2 (dmPGE2). In particular embodiments, the PGE2 compoundthat is administered comprises PGE2 and/or dmPGE2.

In some embodiments, the PGE2 compound that is administered is acompound that attenuates PGE2 catabolism. In some cases, a compound thatattenuates PGE2 catabolism can be a compound, a neutralizing peptide, ora neutralizing antibody that inactivates or blocks15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivates or blocksa prostaglandin transporter, which transports PGE2 inside cells forcatabolism by 15-PGDH. The prostaglandin transporter is also known as2310021C19Rik, MATR1, Matrin F/Q, OATP2A1, PGT, PHOAR2, SLC21A2, solutecarrier organic anion transporter family member 2A1, and SLCO2A1.

In some embodiments, the composition that is administered according tothe methods of the present invention may include a stem-cell inducingmolecule. In some cases, the stem-cell inducing molecule is a PGE2compound as described herein. Other non-limiting examples of stem-cellinducing molecules that may be used herein include oxytocin, betaintegrin activating antibody, rapamycin, SetD7 inhibitors, p38 MAPKinhibitors (such as SB202190 and SB203580), neuregulin, nerve growthfactor (NGF), Hif2alpha inhibitors, basic fibroblast growth factor(bFGF), fibroblast growth factor 4 (FGF4), epidermal growth factor(EGF), Interleukin-1α, Interleukin-13, TNFα, LIF, IL6, interferon gamma,oncostatin M (OSM), ghrelin, and apelin.

In some embodiments, the myotoxin that is administered according tomethods of the present invention is selected from the group consistingof an anesthetic, a divalent cation, venom from snakes, venom fromlizards, venom from bees, and a combination thereof. Suitable divalentcations include but are not limited to Ba²⁺, Sr²⁺, Mg²⁺, Ca²⁺, Mn²⁺,Ni²⁺, Co²⁺, salts thereof, and combinations thereof. In someembodiments, the snake or lizard venom is selected from the groupconsisting of notexin, cardiotoxin, bungarotoxin, and a combinationthereof.

In some embodiments, the anesthetic is selected from the groupconsisting of an amino-amide anesthetic, an amino-ester anesthetic, anda combination thereof. In some cases, the anesthetic is a mild myotoxin.Non-limiting examples of amino-amide anesthetics include bupivacaine,levobupivacaine, articaine, ropivacaine, butanilicaine, carticaine,dibucaine, etidocaine, lidocaine, mepivacaine, prilocaine, andtrimecaine. In some embodiments, the composition comprises a combinationof amino-amide anesthetics.

In some embodiments, the anesthetic is an amino-ester anesthetic. Inparticular embodiments, the amino-ester anesthetic is an aminobenzoicacid ester anesthetic, a benzoic acid ester anesthetic, or a combinationthereof. Non-limiting examples of aminobenzoic acid ester anestheticsinclude benzocaine, butacaine, butamben, chloroprocaine, dimethocaine,lucaine, meprylcaine, metabutethamine, metabutoxycaine, nitracaine,orthocaine, propoxycaine, procaine, proxymetacaine, risocaine, andtetracaine. Non-limiting examples of benzoic acid anesthetics includeamylocaine, cocaine, cyclomethycaine, α-eucaine, β-eucaine, hexylcaine,isobucaine, and piperocaine. In particular embodiments, the compositioncomprises a combination of one or more aminobenzoic acid esteranesthetics and/or one or more benzoic acid ester anesthetics.

Other non-limiting examples of anesthetics that may have mild myotoxiceffects include benzonatate, diperodon, fomocaine, fotocaine,hydroxyprocaine, oxetacaine, oxybuprocaine, paraethoxycaine, phenacaine,piridocaine, pramocaine, primacaine, procainamide, proparacaine,pyrrocaine, quinisocaine, tolycaine, and tropacocaine.

In some embodiments, the composition for administration according tomethods of the present invention comprises a PGE2 compound thatcomprises PGE2 and/or dmPGE2 and a myotoxin that is an anesthetic (e.g.,bupivacaine). In particular embodiments, no anesthetic is administeredto the subject. In some embodiments, a myotoxin that is not ananesthetic is administered to the subject.

The methods provided herein can be used to prevent or treat a musclecondition or disease (e.g., a muscle condition or disease associatedwith muscle damage, injury, or atrophy) in a subject in need thereof.The method can provide prophylactic treatment to a subject who is likelyto experience a muscle condition (e.g., muscle damage, injury, oratrophy). In some embodiments, the subject can have a condition ordisease with possible secondary symptoms that affect muscle. In otherembodiments, the subject has undergone a surgical or therapeuticprocedure or intervention to treat the muscle condition or disease, andthe method disclosed herein is used to prevent or inhibit recurrence orrelapse. In some embodiments, the subject has any one of the conditionsor diseases described herein that affects muscle.

As used herein, the term “treatment” or “treating” encompassesadministration of compounds and/or cells in an appropriate form prior tothe onset of disease symptoms and/or after clinical manifestations, orother manifestations of the condition or disease to reduce diseaseseverity, halt disease progression, or eliminate the disease. The term“prevention of” or “preventing” a disease includes prolonging ordelaying the onset of symptoms of the condition or disease, preferablyin a subject with increased susceptibility to the condition or disease.In some embodiments, treating the subject produces an improvement inmuscle strength and/or muscle coordination.

The methods provided herein may be useful for promoting muscleregeneration in a subject in need thereof, for increasing muscle mass ina subject in need thereof, or both. Regeneration of muscle includesforming new muscle fibers from muscle stem cells, satellite cells,muscle progenitor cells, and any combination thereof. The methods arealso useful for enhancing or augmenting muscle repair, maintenance, orboth. Furthermore, by promoting muscle regeneration, the methodsprovided herein also promote neuromuscular junction establishment andrestoration of muscle contractile function and volume.

In some embodiments, the methods provided herein comprise administeringa composition comprising a PGE2 compound and a myotoxin to a muscle or amuscle cell in vivo. In other embodiments, the methods provided hereincomprise providing to a muscle cell a first composition comprising aPGE2 compound ex vivo, and administering the muscle cell to a muscle invivo. In some cases, the first composition may further comprise amyotoxin. In other cases, the administering the muscle cell to a musclein vivo further comprises administering a myotoxin to the muscle invivo.

In some embodiments, the methods provided herein further compriseadministering a senolytic drug. A senolytic drug is a drug that inducesclearance of senescent cells that produce a senescence-associatedsecretory phenotype. In some cases, a senolytic drug is a drug thattargets a pathway involving BCL-2, BCL-XL, MDM2, p53, p21, serpine(PAI-1&2), HSP-90, PI3Kδ, AKT, HIF1 alpha, ephrin, or a combinationthereof. Examples of a senolytic drug include dasatinib, alvespimycin,geldanamycin, tanespimycin; fisetin, ABT-263, ABT-767, A1331852, andA1155463. In some cases, administering of a senolytic drug is before,during, after, or a combination, administering a composition comprisinga PGE2 compound and a myotoxin.

According to methods of the present invention, compositions andpharmaceutical compositions of the present invention (e.g., comprising acombination of a PGE2 compound and a myotoxin, or comprising a PGE2receptor agonist and optionally a myotoxin) can be administered to asubject experiencing a muscle condition such as muscle injury,degeneration, damage, atrophy, or any combination thereof. In someinstances, the muscle condition is the result of partial or completedenervation. Muscle atrophy can include loss of muscle mass, loss ofmuscle strength, or both. Muscle atrophy may affect any muscle of asubject. In some cases, the subject in need of the compositions,methods, and kits provided herein may be exhibiting or experiencingmuscle loss due to, e.g., age, inactivity, injury, disease, or anycombination thereof.

In some embodiments, compounds can activate muscle cell proliferation,muscle cell differentiation, fusion of muscle cells, or any combinationthereof. In some cases, the muscle tissue may be regenerated. In othercases, muscle function (e.g., muscle mass, muscle strength, musclecontraction, or any combination thereof) may be restored or enhanced. Insome cases, muscle weakness and atrophy may be ameliorated.

The damaged muscle can be any muscle of the body, including but notlimited to, musculi pectoralis complex, latissimus dorsi, teres majorand subscapularis, brachioradialis, biceps, brachialis, pronatorquadratus, pronator teres, flexor carpi radialis, flexor carpi ulnaris,flexor digitorum superficialis, flexor digitorum profundus, flexorpollicis brevis, opponens pollicis, adductor pollicis (e.g., abductorpollicis brevis, abductor pollicis longus), flexor pollicis brevis,iliopsoas, psoas, rectus abdominis, rectus femoris, gluteus maximus,gluteus medius, medial hamstrings, gastrocnemius, lateral hamstring,quadriceps mechanism, adductor longus, adductor brevis, adductor magnus,gastrocnemius medial, gastrocnemius lateral, soleus, tibialis posterior,tibialis anterior, flexor digitorum longus, flexor digitorum brevis,flexor hallucis longus, extensor hallucis longus, hand muscles, armmuscles, foot muscles, leg muscles, chest muscles, stomach muscles, backmuscles, buttock muscles, shoulder muscles, head and neck muscles,facial muscles, oculopharyngeal muscles, and the like. In someinstances, the muscle may be an abductor pollicis brevis muscle.

Subjects in need of muscle regeneration may have musculoskeletalinjuries (e.g., fractures, strains, sprains, acute injuries, overuseinjuries, and the like), post-trauma damages to limbs or face, athleticinjuries, post-fractures in the aged, soft tissue hand injuries, muscleatrophy (e.g., loss of muscle mass), Duchenne muscular dystrophy (DMD),Becker muscular dystrophy, Fukuyama congenital muscular dystrophy(FCMD), limb-girdle muscular dystrophy (LGMD), congenital musculardystrophy, facioscapulohumeral muscular dystrophy (FHMD), myotonicmuscular dystrophy, oculopharyngeal muscular dystrophy, distal musculardystrophy, Emery-Dreifuss muscular dystrophy, myotonia congenita,myotonic dystrophy, other muscular dystrophies, muscle wasting disease,such as cachexia due to cancer, end stage renal disease (ESRD), acquiredimmune deficiency syndrome (AIDS), or chronic obstructive pulmonarydisease (COPD), post-surgical muscle weakness, post-traumatic muscleweakness, sarcopenia, inactivity (e.g., muscle disuse or immobility),urethral sphincter deficiency, urethral sphincter deficiency,neuromuscular disease, and the like.

Non-limiting examples of neuromuscular diseases include, but are notlimited to, acid maltase deficiency, amyotrophic lateral sclerosis,Andersen-Tawil syndrome, Becker muscular dystrophy, Becker myotoniacongenita, Bethlem myopathy, bulbospinal muscular atrophy, carnitinedeficiency, carnitine palmityl transferase deficiency, central coredisease, centronuclear myopathy, Charcot-Marie-Tooth disease, congenitalmuscular dystrophy, congenital myasthenic syndromes, congenital myotonicdystrophy, Cori disease, Debrancher enzyme deficiency, Dejerine-Sottasdisease, dermatomyositis, distal muscular dystrophy, Duchenne musculardystrophy, dystrophia myotonica, Emery-Dreifuss muscular dystrophy,endocrine myopathies, Eulenberg disease, facioscapulohumeral musculardystrophy, tibial distal myopathy, Friedreich's ataxia, Fukuyumacongenital muscular dystrophy, glycogenosis type 10, glycogenosis type11, glycogenosis type 2, glycogenosis type 3, glycogenosis type 5,glycogenosis type 7, glycogenosis type 9, Gowers-Laing distal myopathy,hereditary inclusion-body myositis, hyperthyroid myopathy, hypothyroidmyopathy, inclusion-body myositis, inherited myopathies,integrin-deficient congenital muscular dystrophy, spinal-bulbar muscularatrophy, spinal muscular atrophy, lactate dehydrogenase deficiency,Lambert-Eaton myasthenic syndrome, McArdel disease, merosin-deficientcongenital muscular dystrophy, metabolic diseases of muscle,mitochondrial myopathy, Miyoshi distal myopathy, motor neuron disease,muscle-eye-brain disease, myasthenia gravis, myoadenylate deaminasedeficiency, myofibrillar myopathy, myophosphorylase deficiency, myotoniacongenital, myotonic muscular dystrophy, myotubular myopathy, nemalinemyopathy, Nonaka distal myopathy, oculopharyngeal muscular dystrophy,paramyotonia congenital, Pearson syndrome, periodic paralysis,phosphofructokinase deficiency, phosphoglycerate kinase deficiency,phosphoglycerate mutase deficiency, phosphorylase deficiency,polymyositis, Pompe disease, progressive external ophthalmoplegia,spinal muscular atrophy, Ullrich congenital muscular dystrophy, Welanderdistal myopathy, ZASP-related myopathy, and the like.

Muscle atrophy (e.g., muscle wasting) can be caused by or associatedwith, for example, normal aging (e.g., sarcopenia), geneticabnormalities (e.g., mutations or single nucleotide polymorphisms), poornourishment, poor circulation, loss of hormonal support, disuse of themuscle due to lack of exercise (e.g., bedrest, immobilization of a limbin a cast, etc.), a surgical procedure (e.g., surgical treatment),trauma (e.g., accidental trauma), injury (e.g., accidental injury),aging, damage to the nerve innervating the muscle, poliomyelitis,amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), heartfailure, liver disease, diabetes, obesity, metabolic syndrome,demyelinating diseases (e.g., multiple sclerosis, Charcot-Marie-Toothdisease, Pelizaeus-Merzbacher disease, encephalomyelitis, neuromyelitisoptica, adrenoleukodystrophy, and Guillian-Barre syndrome), denervation,fatigue, exercise-induced muscle fatigue, frailty, neuromusculardisease, weakness, chronic pain, and the like.

In particular embodiments, the muscle condition or disease that isprevented or treated is selected from the group consisting of: traumaticinjury (e.g., acute muscle trauma, acute nerve trauma), acute muscleinjury, acute nerve injury, chronic nerve injury, soft tissue handinjury, carpal tunnel syndrome (CTS), Duchenne muscular dystrophy (DMD),Becker muscular dystrophy, limb girdle muscular dystrophy, amyotrophiclateral sclerosis (ALS), distal muscular dystrophy (DD), inheritedmyopathies, myotonic muscular dystrophy (MDD), mitochondrial myopathies,myotubular myopathy (MM), myasthenia gravis (MG), congestive heartfailure, periodic paralysis, polymyositis, rhabdomyolysis,dermatomyositis, cancer cachexia, AIDS cachexia, cardiac cachexia,stress induced urinary incontinence, sarcopenia, spinal muscularatrophy, fecal sphincter dysfunction, Bell's palsy, rotator cuff injury,spinal cord injury, hip replacement, knee replacement, wrist fracture,diabetic neuropathy, gastroesophageal reflux disease (GERD), obstructivesleep apnea (OSA), pelvic floor disorders (e.g., stress urinaryincontinence, overactive bladder/urinary urgency incontinence, mixedurinary incontinence, pelvic organ prolapse, fecal incontinence),musculoskeletal disorders (e.g., impaired hand function, impaired thumbfunction, impaired foot function), plantar fasciitis, foot drop,disuse-induced muscle atrophy, impaired eyelid function (e.g., eyeliddrooping, impaired blinking, entropion, ectropion), strabismus,nystagmus, and presbyopia. In some instances, the subject has ulnarnerve entrapment (e.g., at the elbow), either with or without musclewasting. Additional examples of suitable conditions may include muscledisorders that affect small isolated muscles that can be regeneratedwith localized transplantation of small numbers of cells, including:atrophy and muscle dysfunction in the face or hand after nerve injury ordirect trauma that does not recover after reinnervation; extraocularmuscle injury causing inability to move the eye and dipoplia seen inGraves' disease, traumatic injury, and progressive externalophthalmoplegia; and urinary and fecal incontinence.

In some embodiments, the subject has received a traumatic injury. Inother embodiments, the muscle condition being treated is a traumaticinjury. In particular embodiments, the traumatic injury comprises blunttrauma or a crush injury. In some instances, the traumatic injurycomprises blunt trauma or a crush injury to a limb (e.g., arm, leg,hand, foot, digit). In some embodiments, the traumatic injury isaccidental. In some embodiments, the PGE2 compound (e.g., PGE2 receptoragonist) is administered immediately after the traumatic injury hasoccurred. In some embodiments, a combination of the PGE2 compound (e.g.,PGE2 receptor agonist) and the myotoxin is administered immediatelyafter the traumatic injury has occurred. In some embodiments, the PGE2compound (e.g., PGE2 receptor agonist) and the myotoxin are administeredsimultaneously to the subject. In some embodiments, the PGE2 compound orthe combination of the PGE2 compound and the myotoxin is administeredwithin about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, or 60 minutes after occurrence of the traumaticinjury. In other embodiments, the PGE2 compound or the combination ofthe PGE2 compound and the myotoxin is administered within about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours of occurrence of the traumatic injury. The PGE2 compound(e.g., PGE2 receptor agonist) or the combination of the PGE2 compoundand the myotoxin can be administered, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore times following occurrence of the traumatic injury.

In some embodiments, a subject who is treated (e.g., for a musclecondition or disease, or prophylactically) according to methods of thepresent invention receives a surgical procedure (e.g., surgicaltreatment). In some embodiments, the surgical procedure is for theprevention, reduction, or repair of a nerve injury. As a non-limitingexample, the nerve injury (e.g., that is surgically treated) can be aperipheral nerve injury. In some instances, a subject who is treated orgiven prophylactic treatment (e.g., for a muscle condition or disease)according to methods of the present invention undergoes a carpal tunnelrelease procedure. In some embodiments, the surgical procedure comprisescutting a muscle, repairing a muscle, or both. As a non-limitingexample, a PGE2 compound (e.g., a PGE2 receptor agonist), or acombination of a PGE2 compound and a myotoxin can be administered inconjunction with a Caesarean section, a hip replacement, or a kneereplacement (e.g., a PGE2 compound, or a combination of a PGE2 compoundand a myotoxin can be administered at the same time that a Caesareansection, hip replacement, or knee replacement is performed). In someembodiments, the methods of the present invention enhance post-operativerecovery. Methods of the present invention can also be used to enhancethe function of small muscles, the strength of small muscles, or both(e.g., hand, facial, oculopharyngeal muscles). When used in conjunctionwith a surgical procedure, methods of the present invention can beperformed, before surgery, during surgery, after surgery, or anycombination thereof. In some embodiments, only a PGE2 compound (e.g., aPGE2 receptor agonist) is administered (e.g., before, at the same time,or after a surgical procedure). In particular embodiments, no anestheticis delivered. As a non-limiting example, in some instances, methods ofthe present invention may eliminate the need for the administration ofmarcaine.

The compositions and pharmaceutical compositions (e.g., comprising aPGE2 compound and a myotoxin, or comprising a PGE2 receptor agonist andoptionally a myotoxin) can be administered topically, orally,intraperitoneally, intramuscularly, intra-arterially, intradermally,subcutaneously, intravenously, intracranially, intrathecally,intraspinally, intralesionally, intranasally, intracerebroventricularly,by inhalation and/or by intracardiac injection. The compositions can beadministered in accordance with an acute regimen (e.g., single orintermittent dosing) or a chronic regimen (e.g., continuous dosing).

When a combination of a PGE2 compound (e.g., a PGE2 receptor agonist)and a myotoxin are administered, the PGE2 compound and the myotoxin canbe administered concomitantly or sequentially. When the PGE2 compoundand the myotoxin are administered sequentially, the PGE2 compound can beadministered first, followed by the myotoxin, or vice versa. In someembodiments, the order of sequential administration alternates orotherwise varies between treatments (e.g., during one treatment, a PGE2compound is administered first, followed by administration of themyotoxin, then during a subsequent treatment the myotoxin isadministered first, followed by the PGE2 compound).

When a PGE2 compound (e.g., a PGE2 receptor agonist) and a myotoxin areadministered sequentially, administration of the compounds can beseparated by some length of time. In some cases, administration of thecompounds is separated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or more minutes. Inother cases, administration of the compounds is separated by about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or more hours. In other cases, administration of the compoundsis separated by about 1, 2, 3, 4, 5, 6, 7, or more days.

In some embodiments, a dose of the PGE2 compound (e.g., PGE2 receptoragonist), the myotoxin, or both, is determined based upon the size of atarget muscle. As a non-limiting example, a dose can comprise about 10ng of the PGE2 compound (e.g., PGE2 receptor agonist), the myotoxin, orboth, when the target muscle is an abductor pollicis brevis muscle(e.g., an abductor pollicis brevis muscle that is of about averagesize). As other non-limiting examples, a dose can comprise about 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 25, 40, 45, 50, or more mg of the PGE2 compound(e.g., PGE2 receptor agonist), the myotoxin, or both, per kg of muscletissue.

In other embodiments, a dose of the PGE2 compound (e.g., PGE2 receptoragonist), the myotoxin, or both, is based on the body weight of thesubject. In particular embodiments, a dose of the PGE2 compound (e.g.,PGE2 receptor agonist), the myotoxin, or both, is about 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 25, 40, 45, 50, or more mg per kg of the subject's body weight.

In some embodiments, the subject is also administered a population ofisolated (or isolated and purified) muscle cells that are eitherautologous or allogeneic to the subject. The cells can be isolated,purified, or both, by any method known to those of skill in the art. Thecells can be a homogenous or heterogeneous population of muscle cells.

The isolated muscle cells can be administered by injection ortransplantation. In some embodiments, compositions, the pharmaceuticalcompositions, or both, described herein (e.g., comprising a PGE2compound and a myotoxin, or comprising a PGE2 receptor agonist andoptionally a myotoxin) and the cells may be administered together orconcomitantly. In other embodiments, the compositions, thepharmaceutical compositions, or both, and the cells may be administeredsequentially. In some cases, the compositions, the pharmaceuticalcompositions, or both, may be administered before the cells. In othercases, the cells may be administered before the compositions, thepharmaceutical compositions, or both. Furthermore, the cells can beadministered before, during, or after a surgical procedure (e.g.,surgical treatment, e.g., for treatment of a nerve injury or a musclecondition or disease).

The population of muscle cells administered to the subject can includeskeletal muscle cells, smooth muscle cells, cardiac muscle cells,embryonic stem cell-derived muscle cells, induced pluripotent stemcell-derived muscle cells, dedifferentiated muscle cells, or anycombinations thereof. Additionally, the muscle cells administered to thesubject can be muscle stem cells, satellite cells, myocytes, myoblasts,myotubes, myofibers, or any combination thereof. The compositions and/orpharmaceutical compositions described herein (e.g., comprising a PGE2compound and a myotoxin, or comprising a PGE2 receptor agonist andoptionally a myotoxin) can be administered to the subject by topical,oral, intraperitoneal, intramuscular, intra-arterial, intradermal,subcutaneous, intravenous, or intracardiac administration. In somecases, the compositions and/or pharmaceutical compositions may beadministered directly to the dysfunctional, injured, damaged and/oratrophied muscle. The compositions and/or pharmaceutical compositionscan be administered in accordance with an acute regimen (e.g., single orintermittent dosing) or a chronic regimen (e.g., continuous dosing).

Satellite cells are small mononuclear progenitor cells that can residewithin muscle tissue. These cells can be induced to proliferate anddifferentiate into muscle cells, and in some instances, fuse to musclefibers. During muscle damage or injury, quiescent satellite cells (e.g.,satellite cells that are not differentiating or undergoing cell divisionat present) and muscle stem cells can be activated to proliferate,and/or migrate out of the muscle stem cell niche. The satellite cellsand muscle stem cells can also differentiate into myocytes, myoblasts,or other muscle cell types.

Methods and protocols for generating muscle cells from embryonic stemcells are described, e.g., in Hwang et al., PLoS One, 2013, 8(8):e72023;and Darabi et al., Cell Stem Cell, 2012, 10(5):610-9. Methods andprotocols for generating muscle cells from induced pluripotent stemcells are described, e.g., in Darabi et al., Cell Stem Cell, 2012,10(5):610-9; Tan et al., PLoS One, 2011; and Mizuno et al., FASEB J.,2010, 24(7):2245-2253.

In some embodiments, muscle cells are obtained by biopsy from a musclesuch as a mature or adult muscle, e.g., quadriceps, gluteus maximus,biceps, triceps, or any muscle from an individual. The muscle can be askeletal muscle, smooth muscle, or cardiac muscle. Detailed descriptionsof methods of isolating smooth muscle stem cells can be found, e.g., inU.S. Pat. No. 8,747,838, and U.S. Patent App. Publ. No. 20070224167.Methods of isolating muscle cells of interest such as muscle stem cellsor satellite cells from muscle tissue are described in detail, forexample, in Blanco-Bose et al., Exp. Cell Res., 2001, 26592:212-220.

Methods for purifying a population of muscle cells of interest, e.g.,muscle stem cells, muscle satellite cells, myocytes, myoblasts,myotubes, and/or myofibers include selecting, isolating or enriching fora cell having a specific cell surface marker or a specific polypeptidethat is expressed on the cell surface of the muscle cell of interest.Useful cell surface markers are described in, e.g., Fukada et al.,Front. Physiol., 2013, 4:317. Cell sorting methods such as flowcytometry, e.g., fluorescence-activated cell sorting (FACS); magneticbead cell separation, e.g., magnetic-activated cell sorting (MACS), andother antibody-based cell sorting methods can be performed to isolate orseparate the muscle cells of interest from other cell types.

The isolated population of muscle cells of interest can be expanded ormultiplied using conventional culture-based methods. Methods for culturemuscle cells are found in, e.g., U.S. Pat. No. 5,324,656. In some cases,the cells may be cultured on a scaffold or gel such as a hydrogel.

In some embodiments, the cells may be stimulated to proliferate byculturing the cells with the PGE2 compound (e.g., PGE2 receptor agonist)and/or myotoxin prior to administering them to the subject. The cellscan be acutely, intermittently or continuously exposed to the compoundduring in vitro culturing. In some cases, the population of muscle cellsmay increase by at least about 1%, at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 100%, at least about200%, at least about 500%, at least about 1000%, or more after culturingwith the PGE2 compound (e.g., PGE2 receptor agonist) and/or myotoxin.

The methods described herein can be used to increase the number ofmuscle fibers by at least about 1%, at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 100%, at least about200%, at least about 500%, at least about 1000%, or more. In someembodiments, the methods can increase the growth of a damaged, injured,atrophied, or degenerated muscle.

In some embodiments, a target muscle may be subjected to mechanicalinjury. As non-limiting examples, mechanical injury can comprisecutting, burning, freezing, needle puncture, exercise (e.g., brief orprolonged exercise), a surgical procedure (e.g., surgical treatment),traumatic injury (e.g., accidental trauma or injury), or a combinationthereof. In some instances, the mechanical injury is before, after, orconcomitant with administration of a PGE2 compound (e.g., PGE2 receptoragonist). In some embodiments, when performed in conjunction withadministration of a PGE2 compound (e.g., PGE2 receptor agonist),mechanical injury acts as a regenerative inducer that stimulates musclecell proliferation, muscle cell growth, muscle cell survival, muscleregeneration, muscle growth, and/or an increase in muscle mass. Inparticular embodiments, mechanical injury acts as a regenerative inducerwhen a myotoxin is not administered to the subject.

In some instances, the mechanical injury is before, after, orconcomitant with administration of a PGE2 receptor agonist. In someinstances, the mechanical injury is before, after, or concomitant withadministration of a myotoxin. In some other instances, the mechanicalinjury is before, after, or concomitant with administration of acombination of a PGE2 compound (e.g., PGE2 receptor agonist) and amyotoxin.

C. Methods of Treating Ocular Disorders or Diseases

In one aspect, the methods may comprise the administration of atherapeutic composition comprising a PGE2 compound and/or a myotoxin, asdescribed herein, to the ocular system of a subject in need thereof. Insome embodiments, the therapeutic composition comprising a PGE2 compoundand/or a myotoxin can be administered to a subject in need thereof totreat an ocular disorder or disease. In other embodiments, thetherapeutic composition comprising a PGE2 compound and/or a myotoxin canbe administered to a subject in need thereof to improve an eye function.In further embodiments, the therapeutic composition comprising a PGE2compound and/or a myotoxin can be administered to a subject in needthereof to enhance the effectiveness of an existing approach to treat anocular disorder or disease, such as cataract surgery or retinal surgery.

Eyelid Function

The eyelids protect the eyes. When the eye blinks, the eyelid spreadsmoisture over the eyes. Blinking also helps move dirt and otherparticles off the surface of the eye. When something approaches the eye,the eyelid closes to protect the eye against injuries. Eyelid functionmay be impaired and result in an ocular disorder or disease. In somecases, the eyelid may droop, resulting in a disorder such as ptosis. Inother cases, the eyelid may turn in or out, resulting in disorders suchas entropion or ectropion. In other cases, the eyelid may have abnormalblinking or twitching, resulting in disorders such as dry eye syndromeor wet eye syndrome (epiphora). In certain aspects, methods are providedherein for the treatment of an ocular disease or disorder due toimpaired eyelid function. In some cases, the method comprisesadministering a therapeutic composition comprising a PGE2 compoundand/or a myotoxin to a subject in need thereof to improve an eyelidfunction. In some cases, an eyelid function may be improved by inducingmuscle regeneration in an eye muscle. The eye muscle may be a musclethat impacts an eyelid function. Non-limiting examples of eye musclesinclude the levator muscle, Muller's muscle or orbicularis. In somecases, the methods may comprise administering a therapeutic compositionof the disclosure (e.g., a PGE2 compound and/or a myotoxin) to an eyemuscle.

Eyelid Drooping

Eyelid drooping is excess sagging of the upper eyelid. In some cases,the edge of the upper eyelid may be lower than it should, also known asptosis. In other cases, there may be excess baggy skin in the uppereyelid, also known as dermatochalasis. In other cases, it may be acombination of ptosis and dermatochalasis. Eyelid drooping may be due tothe weakness of an eyelid muscle. In some cases, the cause of theweakness of the eyelid muscle may be due to the normal aging process, ora result of an injury or a disease. In some cases, eyelid drooping maybe associated with another disorder such as a tumor around or behind theeye, diabetes, Horner syndrome, Myasthenia gravis, stroke, swelling inthe eyelid (e.g., stye). In other cases, eyelid drooping may becongenital. In other cases, eyelid drooping may be due to botoxadministration/exposure.

In some aspects, the methods of the disclosure may involve treatingeyelid drooping. In some cases, the methods may comprise administering atherapeutic composition comprising a PGE2 compound and/or a myotoxin toa subject having, suspected of having, or at risk of developing eyeliddrooping. In some cases, eyelid drooping may include ptosis,dermatochalasis, or both. In some embodiments, treating eyelid droopingmay include treating ptosis, dermatochalasis, or both. In someembodiments, the methods may involve treating eyelid drooping caused byweakness of the eye muscle, such as weakness due to the aging process,or as a result of injury or disease. In some embodiments, the methodsmay involve treating eyelid drooping caused by a tumor around or behindthe eye, Horner syndrome, Myasthenia gravis, stroke, or swelling in theeye (e.g., stye). In some cases, the methods involve administering atherapeutic composition of the disclosure to an eyelid muscle of asubject having eyelid drooping. In some embodiments, the therapeuticcomposition may treat eyelid drooping by inducing muscle regeneration ofan eyelid muscle of a subject. In some cases, the eyelid muscle may bethe levator muscle, Muller's muscle, orbicularis, the frontalis muscle,or any one of the facial muscles. In some cases, the methods may involveadministering a therapeutic composition of the disclosure to any one ofthe levator muscle, Muller's muscle, orbicularis, the frontalis muscle,or the facial muscles.

In some embodiments, the therapeutic composition may be administered incombination with eyelift surgery (e.g., blepharoplasty) to treat eyeliddrooping. In some cases, the therapeutic composition can be administeredbefore surgery, during surgery, after surgery, or any combinationthereof. In other embodiments, the therapeutic composition may beadministered without eyelift surgery to treat eyelid drooping.

In some embodiments, the therapeutic composition (e.g., comprising aPGE2 compound and/or a myotoxin) may be administered by topicaladministration, intradermal administration, intramuscularadministration, or a combination thereof. In some cases, the therapeuticcomposition is administered by intramuscular administration. In somecases, intramuscular administration comprises injection of an eyelidmuscle. The eyelid muscle may include any one of the levator muscle,Muller's muscle, the orbicularis muscle, the frontalis muscle, or thefacial muscles. In some cases, an anesthetic may be administered to aneye of a subject prior to injection of the therapeutic composition. Insome cases, the eyelid muscle may be injected with surgical exposure; inother cases, the eyelid muscle may be injected without surgicalexposure. In some embodiments, a 27-, 28-, 29- or 30-gauge needle may beused to inject the eyelid muscle.

In some embodiments, the methods of the disclosure may involve injectingan eyelid muscle of a subject in need thereof with a volume of about0.01 mL to about 0.15 mL of a therapeutic composition of the disclosure(e.g., comprising a PGE2 compound and/or a myotoxin). In someembodiments, the eyelid muscle may be injected with at least about 0.01mL of a therapeutic composition. In some embodiments, the eyelid musclemay be injected with at most about 0.15 mL of a therapeutic compositionof the disclosure (e.g., comprising a PGE2 compound and/or a myotoxin).In some embodiments, the eyelid muscle may be injected with greater than0.01 mL, greater than 0.02 mL, greater than 0.03 mL, greater than 0.04mL, greater than 0.05 mL, greater than 0.06 mL, greater than 0.07 mL,greater than 0.08 mL, greater than 0.09 mL, greater than 0.10 mL,greater than 0.11 mL, greater than 0.12 mL, greater than 0.13 mL, orgreater than 0.14 mL of a therapeutic composition of the disclosure(e.g., comprising a PGE2 compound and/or a myotoxin).

In some aspects, the effectiveness of a therapeutic composition of thedisclosure to treat eyelid drooping may be determined by conductingtests before administration, after administration, or both. In somecases, the tests may determine how much the eyelid is drooping beforeadministration, after administration, or both. In some cases, the testmay be a slit-lamp examination, a tension test, a visual field test, ora combination thereof. In some embodiments, the dose of the therapeuticcomposition may be adjusted after determining the effectiveness of aprior administration. In some cases, the dose of the PGE2 compound, themyotoxin, or both, may increase. In other cases, the dose of the PGE2compound, the myotoxin, or both, may decrease. In some cases, the doseof the PGE2 compound, the myotoxin, or both, may not change. In someembodiments, the frequency of administration of a therapeuticcomposition of the disclosure (e.g., comprising a PGE2 compound, amyotoxin, or both) may be adjusted after determining the effectivenessof an administration of the therapeutic composition. In some cases, onlya single administration of the therapeutic composition may be needed totreat eyelid drooping. In some cases, two, three, four, five, or morethan five administrations of the therapeutic composition may be needed.In some cases, the frequency of administration may be increased afterdetermining the effectiveness of a prior administration. In other cases,the frequency of administration may be decreased after determining theeffectiveness of a prior administration.

In some embodiments, a subject in need of a therapeutic composition ofthe disclosure may be identified by a test prior to administration ofthe therapeutic composition. In some cases, the test may be aphenylephrine chemical test. In such cases, a positive reaction to thephenylephrine chemical test may identify the subject as a candidate forregeneration of Muller's muscle. In some cases, a subject may be treatedfor eyelid drooping by injecting Muller's muscle with a therapeuticcomposition of the disclosure (e.g., comprising a PGE2 compound, amyotoxin, or both).

In some aspects, methods are provided for treating irregular astigmatismcomprising administering a therapeutic composition of the disclosure(e.g., comprising a PGE2 compound, a myotoxin, or both) to a subject inneed thereof. Irregular astigmatism may be due to scarring of the corneaas a result of eyelid dropping. In some cases, irregular astigmatism maybe treated by treating eyelid drooping as described herein.

Impaired Blinking

Blinking prevents harmful substances from getting into the eye and maybe important in the homeostasis of a healthy ocular tear and cornealepithelial surface. The lacrimal gland produces a lubricating fluid forthe eye. When the eye blinks, the eyelid moves fluid from the lacrimalgland and across the eye. When the eye becomes irritated, the lacrimalgland produces extra fluid to wash out any impurities. The excess fluiddrains through a tear duct and into the nasal cavity. The blinkingfunction of the eye may become impaired, impacting the health of theeye. In some cases, impaired blinking may result in dry eye syndrome, orsymptoms similar to dry eye syndrome. In other cases, impaired blinkingmay result in wet eye syndrome, or symptoms similar to wet eye syndrome.Impaired blinking may be due to many causes including eyelid laxity,lack of eyelid control, and weakness of other eye muscles. Impairedblinking can cause severe damage to the cornea from desiccation and canlead to devastating corneal diseases such as neurotrophic cornea.

In some aspects, methods are provided for treating impaired blinking ina subject in need thereof. In some cases, the methods may compriseadministering a therapeutic composition of the disclosure (e.g.,comprising a PGE2 compound and/or a myotoxin) to a subject having,suspected of having, or at risk of developing impaired blinking. In somecases, the methods may comprise administering a therapeutic compositionof the disclosure to a subject having, suspected of having, or at riskof developing dry eye syndrome. In some embodiments, the therapeuticcomposition may be administered to treat dry eye syndrome associatedwith impaired blinking, with lacrimal gland atrophy, with 7th nervepalsy, or with repeated styes. In some cases, the methods may compriseadministering a therapeutic composition of the disclosure to a subjecthaving, suspected of having, or at risk of developing wet eye syndrome(epiphora or excessive tearing).

In some embodiments, the therapeutic composition may treat impairedblinking, dry eye syndrome, wet eye syndrome, or a combination thereof,by inducing muscle regeneration in an eye muscle of the subject. In somecases, the eye muscle comprises a muscle that impacts blinking. In somecases, the eye muscle may include any one of the orbicularis muscle, themuscle of Riolan, Homer's muscle, the frontalis muscle, or the facialmuscles. The facial muscles may include the occipitofrontalis muscle,the temporoparietalis muscle, the procerus muscle, the nasalis muscle,the depressor septi nasi muscle, the orbicularis oculi muscle, thecorrugator supercilii muscle, the depressor supercilii muscle, theauricular muscles (anterior, superior and posterior), the orbicularisoris muscle, the depressor anguli oris muscle, the risorius, thezygomaticus major muscle, the zygomaticus minor muscle, the levatorlabii superioris, the levator labii superioris alaeque nasi muscle, thedepressor labii inferioris muscle, the levator anguli oris, thebuccinator muscle, or the mentalis. In some cases, impaired blinking,dry eye syndrome, or wet eye syndrome can be treated by administering atherapeutic composition of the disclosure (e.g., comprising a PGE2compound, a myotoxin, or both) to the orbicularis muscle, the muscle ofRiolan, Homer's muscle, the frontalis muscle, any one of the facialmuscles, or a combination thereof. Recent studies have shown theimportance of muscle stem cells (MuSCs) in stimulating neuromuscularjunctions in denervated muscles (Liu et al., 2015), although untilrecently improving the recovery of muscle function following denervationremained an unsolved problem. A solution to this problem lies in theability to reverse or prevent denervation atrophy by stimulating andaugmenting MuSCs that are already present in the muscles.

In some embodiments, methods of treating impaired blinking, dry eyesyndrome, or wet eye syndrome may include administering a therapeuticcomposition of the disclosure (e.g., comprising a PGE2 compound, and/ora myotoxin) to a subject in need thereof by topical administration,intradermal administration, intramuscular administration, or acombination thereof. In some cases, the therapeutic composition may beadministered by intramuscular administration. In some cases, theintramuscular administration may comprise injection of an eye muscle.The eye muscle may include any one of the orbicularis muscle, the muscleof Riolan, Homer's muscle, the frontalis muscle, or the facial mucles.Recent studies have shown the importance of muscle stem cells (MuSCs) instimulating neuromuscular junctions in denervated muscles, althoughuntil recently improving the recovery of muscle function followingdenervation remained an unsolved problem. A solution to this problemlies in the ability to reverse or prevent denervation atrophy bystimulating and augmenting MuSCs that are already present in themuscles.

In some embodiments, the methods may comprise administering atherapeutic composition of the disclosure to a muscle that impacts afunction of the lacrimal gland to treat impaired blinking associatedwith lacrimal gland dysfunction, such as lacrimal gland atrophy. In somecases, an anesthetic may be administered to an eye of the prior toinjection of the therapeutic composition. In some cases, the eyelidmuscle may be injected with surgical exposure; in other cases, theeyelid muscle may be injected without surgical exposure. In someembodiments, a 27-, 28-, 29- or 30-gauge needle may be used to injectthe eyelid muscle.

In some embodiments, methods of treating impaired blinking, dry eyesyndrome, wet eye syndrome, or a combination thereof may includeadministering (e.g., intramuscular injection) a therapeutic compositionof the disclosure to an eye muscle of a subject in need thereof, in avolume of about 0.01 mL to about 0.15 mL. In some embodiments, the eyemuscle may be injected with at least about 0.01 mL of a therapeuticcomposition of the disclosure. In some embodiments, the eye muscle maybe injected with at most about 0.15 mL of a therapeutic composition ofthe disclosure. In some embodiments, the eye muscle may be injected withgreater than 0.01 mL, greater than 0.02 mL, greater than 0.03 mL,greater than 0.04 mL, greater than 0.05 mL, greater than 0.06 mL,greater than 0.07 mL, greater than 0.08 mL, greater than 0.09 mL,greater than 0.10 mL, greater than 0.11 mL, greater than 0.12 mL,greater than 0.13 mL, or greater than 0.14 mL of a therapeuticcomposition of the disclosure.

In some aspects, the effectiveness of an administration of a therapeuticcomposition may be determined by conducting tests before administration,after administration, or both. For treatment of wet eye syndrome, afluorescein and Lissamine green staining test, optical coherencetomography of the tear film (OCT), or both, may be conducted. Fortreatment of dry eye syndrome, a visual acuity measurement, a slit lampexam, measurement of tear film break-up time (TBUT), measurement of rateof tear production (Schirmer test), measurement of concentration oftears (osmolality), or a combination thereof may be conducted. In somecases, the test may involve measuring levels of inflammatory or growthfactor molecules including, without limitation, MMP-9, lactoferrin, andNGF-1.

In some embodiments, a dose of the therapeutic composition may beadjusted after determining the effectiveness of a prior administration.In some cases, the dose of the PGE2 compound, the myotoxin, or both, mayincrease. In other cases, the dose of the PGE2 compound, the myotoxin,or both may decrease. In some cases, the dose of the PGE2 compound, themyotoxin, or both may not change. In some embodiments, the frequency ofadministration of a therapeutic composition of the disclosure (e.g.,comprising a PGE2 compound and/or a myotoxin) may be adjusted afterdetermining the effectiveness of a prior administration of thetherapeutic composition. In some cases, only a single administration ofthe therapeutic composition may be needed to treat impaired blinking,dry eye syndrome, or wet eye syndrome. In some cases, two, three, four,five, or more than five administrations of the therapeutic compositionmay be needed to treat impaired blinking, dry eye syndrome, or wet eyesyndrome. In some cases, the frequency of administration may beincreased after determining the effectiveness of a prior administration.In other cases, the frequency of administration may be decreased afterdetermining the effectiveness of a prior administration.

Entropion and Ectropion

The eyelid protects the eye from foreign objects. In some cases, theeyelid does not lie properly on the eye. For example, entropion is theturning in of an edge of an eyelid. In some cases, it causes the lashesof the eye to rub against the eye. This can result in excessive tearing,eye discomfort, eye pain, eye irritation, eye redness, and in someextreme cases, cornea damage and decreased vision. Causes of entropionmay include weakening of eye muscles, especially the muscles in thelower part of the eye. Ectropion is the turning out of the eyelid sothat the inner surface is exposed. In some cases, ectropian may causedry, painful eyes, excessive tearing of the eye (epiphora), chronicconjunctivitis, keratitis, eye redness, or a combination thereof. Causesof ectropion may be due to weakening of the eyelid due to the agingprocess, facial palsy, and the like.

In some aspects, methods are provided for the treatment of entropion,ectropion, or both. In some cases, the methods may compriseadministering a therapeutic composition of the disclosure (e.g.,comprising a PGE2 compound and/or a myotoxin) to a subject having,suspected of having, or at risk of developing entropion or ectropion. Insome embodiments, the methods of treating entropion or ectropion mayinvolve administering a therapeutic composition of the disclosure to aneye muscle of a subject in need thereof. In some cases, the eyelidmuscle may be the orbicularis, the frontalis muscles, or any of thefacial muscles. The facial muscles may include the occipitofrontalismuscle, the temporoparietalis muscle, the procerus muscle, the nasalismuscle, the depressor septi nasi muscle, the orbicularis oculi muscle,the corrugator supercilii muscle, the depressor supercilii muscle, theauricular muscles (anterior, superior and posterior), the orbicularisoris muscle, the depressor anguli oris muscle, the risorius, thezygomaticus major muscle, the zygomaticus minor muscle, the levatorlabii superioris, the levator labii superioris alaeque nasi muscle, thedepressor labii inferioris muscle, the levator anguli oris, thebuccinator muscle, or the mentalis.

In some embodiments, the therapeutic composition may be administered incombination with eyelid surgery (e.g., lateral tarsal strip procedure).In those embodiments, the therapeutic composition can be administeredbefore surgery, during surgery, after surgery, or any combinationthereof. In other embodiments, the therapeutic composition may beadministered without eye surgery.

In some embodiments, the methods provided herein may includeadministering a therapeutic composition of the disclosure (e.g.,comprising a PGE2 compound and/or a myotoxin) to a subject in needthereof by topical administration, intradermal administration,intramuscular administration, or a combination thereof. In some cases,the therapeutic composition may be administered by intramuscularadministration. In some cases, the intramuscular administrationcomprises injection of an eye muscle. In some cases, the eye muscle maycomprise any of the orbicularis muscle, the frontalis muscle, or thefacial muscles. In some cases, an anesthetic may be administered to aneye of a subject in need prior to injection. In some cases, the eyemuscle may be injected with surgical exposure; in other cases, the eyemuscle may be injected without surgical exposure. In some embodiments, a27-, 28-, 29- or 30-gauge needle may be used to inject the eye muscle.

In some embodiments, methods of treating ectropion, entropion, or bothmay include administering (e.g., intramuscular injection) a therapeuticcomposition of the disclosure to an eye muscle of a subject in needthereof, in a volume of about 0.01 mL to about 0.15 mL. In someembodiments, the eye muscle may be injected with at least about 0.01 mLof a therapeutic composition of the disclosure. In some embodiments, theeye muscle may be injected with at most about 0.15 mL of a therapeuticcomposition of the disclosure. In some embodiments, the eye muscle isinjected with greater than 0.01 mL, greater than 0.02 mL, greater than0.03 mL, greater than 0.04 mL, greater than 0.05 mL, greater than 0.06mL, greater than 0.07 mL, greater than 0.08 mL, greater than 0.09 mL,greater than 0.10 mL, greater than 0.11 mL, greater than 0.12 mL,greater than 0.13 mL, or greater than 0.14 mL of a therapeuticcomposition of the disclosure.

In some aspects, the effectiveness of an administration of a therapeuticcomposition of the disclosure (e.g., to treat entropion, ectropion, orboth) may be determined by conducting tests before administration, afteradministration, or both. In some cases, the tests may include anexamination of the eye, the eyelid, or a combination thereof. In someembodiments, the dose of the therapeutic composition may be adjustedafter determining the effectiveness of a prior administration. In somecases, the dose of the PGE2 compound, the myotoxin, or both may beincreased. In other cases, the dose of the PGE2 compound, the myotoxin,or both may be decreased. In some cases, the dose of the PGE2 compound,the myotoxin, or both may not change. In some embodiments, the frequencyof administration of a therapeutic composition of the disclosure (e.g.,comprising a PGE2 compound and/or a myotoxin) may be adjusted afterdetermining the effectiveness of an administration of the therapeuticcomposition (e.g., to treat ectropion, entropion, or both). In somecases, only a single administration of the therapeutic composition maybe needed to treat entropion or ectropion. In some cases, two, three,four, five, or more than five administrations of the therapeuticcomposition may be needed to treat entropion or ectropion. In somecases, the frequency of administration may be increased afterdetermining the effectiveness of a prior administration. In other cases,the frequency of administration may be decreased after determining theeffectiveness of a prior administration.

Extraocular Muscles

The extraocular muscles comprise six muscles that control movement ofthe eye (lateral rectus, medial rectus, superior rectus, inferiorrectus, superior oblique, and inferior oblique) and one muscle (levatorpalpebrae) that controls eyelid elevation. Damage or injury to,weakening of, or improper innervation of any one of these extraocularmuscles can result in an ocular disorder or disease, and reduced visualfunction. In some aspects, methods are provided for treating an oculardisorder or disease by administering a therapeutic composition of thedisclosure (e.g., comprising a PGE2 compound and/or a myotoxin). In somecases, the therapeutic composition may be administered to at least oneextraocular muscle, e.g., lateral rectus, medial rectus, superiorrectus, inferior rectus, superior oblique, inferior oblique, or levatorpalpebrae. In some cases, the methods may comprise administering atherapeutic composition of the disclosure to an extraocular muscle of asubject in need thereof to induce muscle regeneration.

Strabismus

Strabismus is an ocular disorder in which both eyes do not line up inthe same direction. As a result, the eyes do not look at the same objectat the same time. The condition is more commonly known as “crossedeyes”. Six different extraocular muscles (lateral rectus, medial rectus,superior rectus, inferior rectus, superior oblique and inferior oblique)surround each eye and work together to allow both eyes to focus on thesame object. In a patient having strabismus, these muscles do not worktogether. As a result, one eye looks at one object and the other eyeturns in a different direction to focus on another object. In manycases, the cause of strabismus is unknown. In some cases, eyemisalignment is observed at birth or shortly afterwards (congenitalstrabismus). In some cases, disorders associated with strabismus inchildren may include, without limitation, Apery Syndrome, CerebralPalsy, Congenital Rubella, Hemangioma, Incontinentia Pigmenti Syndrome,Noonan Syndrome, Prader-Willi Syndrome, Retinopathy of Prematurity,Retinoblastoma, Traumatic Brain Injury and Trisomy 18. Strabismus candevelop in adults and may be due to many different causes including,without limitation, botulism, diabetes, Graves Disease, Guillain-BarreSyndrome, injury to the eye, shellfish poisoning, stroke, traumaticbrain injury and vision loss from an eye disease or injury.

In some aspects, methods are provided for treating strabismus orcongenital strabismus. In some cases, the methods may compriseadministering a therapeutic composition of the disclosure (e.g., a PGE2compound and/or a myotoxin) to a subject having, suspected of having, orat risk of developing strabismus or congenital strabismus. In someembodiments, the therapeutic composition may treat strabismus orcongenital strabismus by inducing muscle regeneration in at least one ofthe extraocular muscles, e.g., lateral rectus, medial rectus, superiorrectus, inferior rectus, superior oblique, and inferior oblique. In somecases, the methods may comprise administering a therapeutic compositionof the disclosure to any of the lateral rectus muscle, the medial rectusmuscle, the superior rectus muscle, the inferior rectus muscle, thesuperior oblique muscle, and the inferior oblique muscle. In some cases,each eye muscle may have a width of about 5 mm to about 7 mm, a lengthof about 10 mm, and a thickness of less than or equal to 1 mm.

In some embodiments, a therapeutic composition of the disclosure may beadministered in combination with eye muscle surgery to treat strabismusor congenital strabismus. In some cases, the therapeutic composition canbe administered before surgery, during surgery, after surgery, or anycombination thereof. In other cases, the therapeutic composition may beadministered without eye muscle surgery to treat strabismus orcongenital strabismus.

In some embodiments, a therapeutic composition of the disclosure (e.g.,comprising a PGE2 compound and/or a myotoxin) may be administered byinjection of an extraocular muscle (e.g., lateral rectus, medial rectus,superior rectus, inferior rectus, superior oblique, and inferioroblique) for the treatment of strabismus or congenital strabismus. Inother cases, a therapeutic composition of the disclosure may beadministered via slow drug release in a drug releasing depot, by genetherapy methods that may include a cell matrix depot that produces acomposition of the disclosure (e.g., a PGE2 compound, a myotoxin), or apolymeric implant placed near or adjacent to the muscles. In anexemplary embodiment, a local anesthetic, an ocular decongestant, orboth, may be administered to an eye of a subject in need thereof priorto injection of the extraocular muscle. In some cases, the extraocularmuscle may be injected with surgical exposure; in other cases, theextraocular muscle may be injected without surgical exposure. In someembodiments, the extraocular muscle may be injected withelectromyographic guidance; in other embodiments, the extraocular musclemay be injected without electromyographic guidance. In some embodiments,a 27-, 28-, 29- or 30-gauge needle may be used to inject the extraocularmuscle. To correct the misalignment of the eyes, one or more extraocularmuscles can be injected as needed. In some embodiments, extraocularmuscles in both eyes can be injected to correct the misalignment.

In some embodiments, methods of treating strabismus may includeadministering (e.g., intramuscular injection) a therapeutic compositionof the disclosure to an extraocular eye muscle of a subject in needthereof, in a volume of about 0.05 mL to about 0.15 mL. In someembodiments, the extraocular muscle may be injected with at least about0.05 mL of the therapeutic composition. In some embodiments, theextraocular muscle may be injected with at most about 0.15 mL of thetherapeutic composition. In some embodiments, the extraocular muscle maybe injected with greater than 0.05 mL, greater than 0.06 mL, greaterthan 0.07 mL, greater than 0.08 mL, greater than 0.09 mL, greater than0.10 mL, greater than 0.11 mL, greater than 0.12 mL, greater than 0.13mL, or greater than 0.14 mL of the therapeutic composition.

In some aspects, the effectiveness of an administration of a therapeuticcomposition of the disclosure (e.g., to treat strabismus) may bedetermined by conducting tests before administration, afteradministration, or both. In some cases, the tests may determine how muchthe eyes are out of alignment. In some embodiments, a corneal lightreflex test, a cover/uncover test, a retinal exam, an ophthalmic exam,visual acuity, or a combination thereof, may be conducted. In somecases, the effectiveness of an administration of the therapeuticcomposition can be determined by changes in the alignment of the eyesbefore administration and after administration. In some embodiments, thedose of the therapeutic composition may be adjusted after determiningthe effectiveness of a prior administration. In some cases, the dose ofthe PGE2 compound, the myotoxin, or both, may be increased. In othercases, the dose of the PGE2 compound, the myotoxin, or both may bedecreased. In some cases, the dose of the PGE2 compound, the myotoxin,or both, may not be changed.

In some embodiments, the frequency of administration of a therapeuticcomposition of the disclosure (e.g., comprising a PGE2 compound and/or amyotoxin) may be adjusted after determining the effectiveness of anadministration of the therapeutic composition (e.g., to treatstrabismus). In some cases, only a single administration of thetherapeutic composition may be needed to treat strabismus. In somecases, two, three, four, five, or more than five administrations of thetherapeutic composition may be needed. In some cases, the frequency ofadministration may be increased after determining the effectiveness of aprior administration. In other cases, the frequency of administrationmay be decreased after determining the effectiveness of a prioradministration.

Nystagmus

Nystagmus, or eye tremor, is a term used to describe fast,uncontrollable movement of the eyes. The movement may be from side toside (horizontal nystagmus); up and down (vertical nystagmus), or rotary(rotary or torsional nystagmus). In some cases, the movement may be inone eye. In other cases, the movement may be in both eyes. In somecases, nystagmus may be present at birth (Infantile Nystagmus Syndrome(INS)). In some cases, nystagmus may be caused by a congenital diseaseof the eye. In other cases, nystagmus may be acquired through a varietyof causes including intake of certain drugs or medications (e.g.,phenytoin), excessive alcohol, a sedating medicine that can impair afunction of the labyrinth, head injury, an inner ear disorder (e.g.,labyrinthis or Meniere disease), stroke, and thiamine or vitamin B12deficiency. In some cases, eye tremors may be secondary to otherdisorders such as Parkinson's disease.

In some aspects, methods are provided for treating nystagmus. In somecases, nystagmus is horizontal nystagmus, vertical nystagmus, rotary ortorsional nystagmus, or any combination thereof. In some cases,nystagmus is Infantile Nystagmus Syndrome. In some cases, the methodscomprise a therapeutic composition of the disclosure (e.g., comprising aPGE2 compound and/or a myotoxin) to a subject having, suspected ofhaving, or at risk of developing nystagmus. In some embodiments, thetherapeutic composition may treat nystagmus by inducing muscleregeneration in at least one of the extraocular muscles, e.g., lateralrectus, medial rectus, superior rectus, inferior rectus, superioroblique, and inferior oblique. In some cases, the therapeuticcomposition may be administered to at least one of the extraocularmuscles, e.g., lateral rectus, medial rectus, superior rectus, inferiorrectus, superior oblique, and inferior oblique, for the treatment ofnystagmus.

Iris

Located between the cornea and the lens, the iris comprises a sphinctermuscle (sphincter pupillae) and dilator muscles (dilator pupillae). Theround, central opening of the iris is called the pupil. The irismodulates the size of the pupil to control how much light comes into theeye. Impairment of the iris can result in impaired visual function.

In some aspects, methods are provided for treating impaired visualfunction. In some cases, the methods may comprise administering atherapeutic composition of the disclosure (e.g., comprising a PGE2compound and/or a myotoxin), to a subject having, suspected of having,or at risk of developing impaired visual function. In some embodiments,the therapeutic composition may treat impaired visual function byinducing muscle regeneration of an iris muscle. The iris muscle may bethe sphincter muscle, the dilator muscle, or both. In some cases, thetherapeutic composition may be administered topically, intradermally, orintraocularly to a subject in need thereof. In some cases, thetherapeutic composition may be administered by intramuscularadministration to a muscle of the iris (e.g., the sphincter muscle, thedilator muscle). In some aspects, the effectiveness of the treatment maybe determined by observing changes in light sensitivity beforeadministration, after administration, or both.

Ciliary Muscle and Other Intraocular Muscles

The ciliary body is a circular structure that contains the ciliarymuscle. The ciliary muscle changes the shape of the lens when the eyefocuses on a near object in a process called accommodation. Presbyopiais a condition in which the lens of the eye loses its ability to focus,making it hard to see objects up close. Presbyopia is thought to be anatural part of the aging process. One approach to treating presbyopiamay be to induce muscle regeneration of the ciliary muscle. Accordingly,provided herein are methods of treating presbyopia. In some cases, themethods may comprise administering a therapeutic composition of thedisclosure (e.g., comprising a PGE2 compound and/or a myotoxin) to asubject having, suspected of having, or at risk of developingpresbyopia. In some embodiments, the therapeutic composition may treatpresbyopia by inducing muscle regeneration of the ciliary muscle. Insome aspects, the effectiveness of the treatment can be determined byperforming a reading test before administration, after administration,or both.

In some aspects, methods are provided for treating myopia, or tomodulate regression of myopia. In some cases, the methods may compriseadministering a therapeutic composition of the disclosure (e.g.,comprising a PGE2 compound and/or a myotoxin) to a subject having,suspected of having, or at risk of developing myopia, or to a subject tomodulate regression of myopia. In some cases, the therapeuticcomposition may be administered to an eye muscle, such as the ciliarymuscle, a muscle in the sclera, a muscle around the sclera, or anintraocular muscle.

Oculopharyngeal Muscular Dystrophy

Oculopharyngeal muscular dystrophy is a genetic disorder characterizedby slowly progressing muscle disease (myopathy) affecting the muscles ofthe upper eyelids and the throat. Onset is typically during adulthood,most often between 40 and 60 years of age. Symptoms may include, withoutlimitation: eyelid drooping (ptosis), arm and leg weakness, anddifficulty swallowing (dysphagia).

In some aspects, methods are provided for treating oculopharyngealmuscular dystrophy. In some cases, the methods may compriseadministering a therapeutic composition of the disclosure (e.g.,comprising a PGE2 compound and/or a myotoxin) to a subject having,suspected of having, or at risk of developing oculopharyngeal musculardystrophy. In some cases, treating oculopharyngeal muscular dystrophymay involve administering the therapeutic composition to the muscles ofthe upper eyelid, the muscles of the throat, or both.

D. Methods of Treating Musculoskeletal Disorders

Provided herein are applications of therapeutic compositions of thedisclosure (e.g., comprising a PGE2 compound and/or a myotoxin) to themusculoskeletal system. In some embodiments, the therapeutic compositioncan be administered to a subject in need thereof to treat amusculoskeletal disorder. In some cases, the musculoskeletal disorder isa muscle disorder. In other embodiments, the therapeutic composition canbe administered to a subject in need thereof to improve a function ofthe musculoskeletal system. In further embodiments, the therapeuticcomposition can be administered to a subject in need thereof to augmenteffectiveness of an existing treatment of a disorder or disease of themusculoskeletal system such as carpometacarpal arthroplasty.

Impaired function of the musculoskeletal system may be due to differentfactors. In some cases, the muscle disorder may be due to aging. Forexample, sarcopenia is the degenerative loss of skeletal muscle mass,varying from 0.5% to 1% muscle loss per year after the age of 50, and isassociated with aging. In other cases, the muscle disorder may be due todisuse of the affected muscle. In some cases, disuse may be due toimmobilization (e.g., a splint, a cast). In other cases, disuse may bedue to pain (e.g., arthritis). In some cases, the muscle disorder may bemuscle atrophy as a result of denervation. Diseases that affect lowermotor neurons may impair innervation of myofibers resulting in muscleatrophy. In other cases, the muscle disorder may be due to metabolicreasons, such as glucocorticoid-induced muscle atrophy. For example,excess alcohol intake can result in alcoholic myopathy. In anotherexample, chronic diabetes mellitus can damage the nerves that innervatethe hands and feet, resulting in diabetic amyotrophy. Other causes ofimpaired muscle function may include trauma, and muscle-relateddegenerative diseases. Provided herein are methods of treating a muscledisorder due to any of the causes described herein. In some cases, themethods comprise administering a therapeutic composition of thedisclosure (e.g., comprising a PGE2 compound and/or a myotoxin).

Aging Hand

One of the most common changes in aging skeletal muscle in the body is amajor reduction in muscle mass ranging from 25% to 45%, which issometimes described as “sarcopenia of old age”. There are 11 intrinsicmuscles and 15 extrinsic muscles with direct functional roles in thehand. Extrinsic and intrinsic hand muscles produce the force requiredfor gripping objects (grip force). After 60 years of age there is arapid decline in hand-grip strength, by as much as 20-25%. This isaccompanied by a substantial loss of muscle fibers and decreasedmuscle-fiber length, particularly in the thenar muscle group, andcontributes an important role in reduction of action potential. Thethumb intrinsic musculature constitutes approximately 40% of the totalintrinsic musculature of the hand. Three of the main muscles, obliqueadductor pollicis, opponens pollicis, and flexor pollicis brevis, playimportant roles in stabilizing the thumb during strong pinch grips ofobjects, and these movements commonly show age-related dysfunction. Thecontractile capacity of the thenar muscle in elderly people has beenassessed by tetanic stimulation of the median nerve. The higher musclefatigue resistance in elderly adults has been attributed to differencesin both the Peripheral Nervous System and Central Nervous System. Thereis a significant reduction in both action potentials and in the numberof viable motor units associated with the hand muscles in the elderly.

In some aspects, methods are provided for treating impaired handfunction. In some cases, the methods may comprise administering atherapeutic composition of the disclosure (e.g., a PGE2 compound and/ora myotoxin) to a subject in need thereof. In some embodiments, thesubject is greater than 50 years old, greater than 55 years old, greaterthan 60 years old, greater than 65 years, or greater than 70 years old.In some embodiments, the impaired hand function may be a result ofaging. In some embodiments, the therapeutic composition may treat theimpaired hand function by inducing muscle regeneration in a hand muscleof the subject. In some cases, the hand muscle may be an intrinsicmuscle. An intrinsic muscle may include any of the following, withoutlimitation: the three thenar muscles, the three hypothenar muscles, theinterossei muscles, the lumbrical muscles, the palmaris brevis, and theadductor pollicis. In some cases, the hand muscle is an extrinsicmuscle. An extrinsic muscle may include any of the following, withoutlimitation: abductor pollicis longus, extensor pollicis brevis, flexorpollicis longus, flexor carpi radialis, flexor digitorum profundus, fourflexor digitorum superficialis, flexor carpi ulnaris, extensor carpiradialis longus, extensor carpi radialis brevis, extensor indicis,extensor digitorum communis, extensor digiti minimi, and extensor carpiulnaris.

In some embodiments, the therapeutic composition may be administered incombination with hand surgery (e.g., closed reduction and fixationsurgery, tendon repair, nerve repair, surgical drainage and/ordebridement, or joint replacement). In such cases, the therapeuticcomposition can be administered before surgery, during surgery, aftersurgery, or any combination thereof. In other embodiments, thetherapeutic composition may be administered without hand surgery.

In some embodiments, a therapeutic composition of the disclosure (e.g.,comprising a PGE2 compound and/or a myotoxin) may be administered bytopical administration, intradermal administration, intramuscularadministration, or a combination thereof. In some cases, the therapeuticcomposition may be administered by intramuscular administration. In somecases, the intramuscular administration may comprise injection of a handmuscle. The hand muscle may include any one of the following: thenarmuscle, a hypothenar muscle, an interossei muscle, a lumbrical muscle,palmaris brevis, adductor pollicis abductor pollicis longus, flexorpollicis longus, flexor carpi radialis, flexor digitorum profundus, fourflexor digitorum superficialis, flexor carpi ulnaris, extensor pollicisbrevis, extensor carpi radialis longus, extensor carpi radialis brevis,extensor indicis, extensor digitorum communis, extensor digiti minimi,or extensor carpi ulnaris. In some cases, an anesthetic may beadministered to a hand of a subject prior to injection of a therapeuticcomposition. In some cases, the hand muscle may be injected withsurgical exposure; in other cases, the hand muscle may be injectedwithout surgical exposure. In some embodiments, a 20-, 21-, 22-, 23-,24- or 25-gauge needle may be used to inject the hand muscle.

In some embodiments, methods of treating impaired hand function mayinclude administering (e.g., intramuscular injection) a therapeuticcomposition of the disclosure to a hand muscle of a subject in needthereof, in a volume of about 0.01 mL to about 0.15 mL. In someembodiments, the hand muscle is injected with at least about 0.01 mL ofthe therapeutic composition. In some embodiments, the hand muscle isinjected with at most about 0.15 mL of the therapeutic composition. Insome embodiments, the hand muscle is injected with greater than 0.01 mL,greater than 0.02 mL, greater than 0.03 mL, greater than 0.04 mL,greater than 0.05 mL, greater than 0.06 mL, greater than 0.07 mL,greater than 0.08 mL, greater than 0.09 mL, greater than 0.10 mL,greater than 0.11 mL, greater than 0.12 mL, greater than 0.13 mL, orgreater than 0.14 mL of the therapeutic composition.

In some aspects, the effectiveness of an administration of a therapeuticcomposition of the disclosure (e.g., to treat impaired hand function)may be determined by conducting tests before administration, afteradministration, or both. In some cases, the test may measure a handfunction before administration, after administration, or both. In somecases, the test may be a maximum pinch force test, a steadiness pinchforce test, a pegboard test, a two-point discrimination test, aprecision pinch steadiness test, a handgrip force test, or a combinationthereof.

In some embodiments, the dose of a therapeutic composition of thedisclosure may be adjusted after determining the effectiveness of aprior administration (e.g., to treat impaired hand function). In somecases, the dose of the PGE2 compound, the myotoxin, or both may beincreased. In other cases, the dose of the PGE2 compound, the myotoxin,or both, may be decreased. In some cases, the dose of the PGE2 compound,the myotoxin, or both may not be changed.

In some embodiments, the frequency of administration of a therapeuticcomposition of the disclosure (e.g., comprising a PGE2 compound and/or amyotoxin) may be adjusted after determining the effectiveness of anadministration of the therapeutic composition. In some cases, only asingle administration of the therapeutic composition may be needed totreat impaired hand function. In some cases, two, three, four, five, ormore than five administrations of the therapeutic composition may beneeded to treat impaired hand function. In some cases, the frequency ofadministration may be increased after determining the effectiveness of aprior administration. In other cases, the frequency of administrationmay be decreased after determining the effectiveness of a prioradministration.

In some embodiments, a subject in need thereof may be identified by atest prior to administration of the therapeutic composition. In somecases, the test may be a maximum pinch force test, a steadiness pinchforce test, a pegboard test, a two-point discrimination test, aprecision pinch steadiness test, a handgrip force test, or a combinationthereof.

Thenar Atrophy

Compression peripheral nerve injuries (PNI) are a category of nerveinjury caused by constriction of the nerve. Carpal tunnel syndrome (CTS)is the most common peripheral compression neuropathy, resulting frommedian nerve compression at the wrist. Symptoms of carpal tunnelsyndrome may include sensory impairments (e.g., numbness andparesthesias) and motor deficits in the abductor pollicis brevis (APB),opponens pollicis, and the superficial belly of the flexor pollicisbrevis, which are the intrinsic median-innervated thenar muscles.Prolonged compression of the median nerve potentially disturbs motorfunction of the thenar muscles, and in patients with severe carpaltunnel syndrome, atrophy of the thenar muscles may result. For example,severe CTS results in the denervation and atrophy of the APB muscle. TheAPB muscle brings the thumb out of the plane of the palm and is integralto many fine motor activities. The surgical treatment of CTS is torelease the band constricting the median nerve. This allows forregeneration of the motor nerve and potential recovery of the muscle.Unfortunately, many of those with severe CTS have poor functionalrecovery even after the nerve has been released. Restoring function tothese specific muscle groups that control the hand grip can increaseindependence and overall quality of life.

In some aspects, methods are provided for the treatment of impairedthumb function. In some cases, the methods comprise administering atherapeutic composition of the disclosure (e.g., comprising a PGE2compound and/or a myotoxin) to a subject having, suspected of having, orat risk of developing impaired thumb function. In some embodiments, theimpaired thumb function is due to thenar atrophy. In some embodiments,the therapeutic composition may treat impaired thumb function byinducing muscle regeneration in a hand muscle of a subject. In somecases, the therapeutic composition in administered to a hand muscle ofthe subject for the treatment of impaired thumb function. In some cases,the hand muscle is the abductor pollicis brevis (APB), the opponenspollicis, or flexor pollicis brevis. In some cases, the hand muscle isthe abductor pollicis brevis.

In some embodiments, a therapeutic composition of the disclosure mayadministered in combination with hand surgery to treat impaired thumbfunction. In some cases, the hand surgery may be carpel tunnel syndromesurgery. In some cases, the therapeutic composition can be administeredbefore surgery, during surgery, after surgery, or any combinationthereof. In other embodiments, the therapeutic composition may beadministered without hand surgery.

In some embodiments, a therapeutic composition of the disclosure (e.g.,comprising a PGE2 compound and/or a myotoxin) may be administered bytopical administration, intradermal administration, intramuscularadministration, or a combination thereof for the treatment of impairedthumb function. In some cases, the therapeutic composition may beadministered by intramuscular administration. In some cases, theintramuscular administration comprises injection of a hand muscle. Insome cases, the hand muscle may comprise a thenar muscle. In some cases,the hand muscle may comprise the abductor pollicis brevis (APB), theopponens pollicis, or flexor pollicis brevis. In some cases, the handmuscle may be the abductor pollicis brevis. In some cases, an anestheticmay be administered to a hand of a subject in need thereof prior toinjection of a therapeutic composition of the disclosure. In some cases,the hand muscle may be injected with surgical exposure; in other cases,the hand muscle may be injected without surgical exposure. In someembodiments, a 20-, 21-, 22-, 23-, 24- or 25-gauge needle may be used toinject the hand muscle.

In some aspects, methods of treating impaired thumb function may includeadministering (e.g., intramuscular injection) a therapeutic compositionof the disclosure to a hand muscle of a subject in need thereof, in avolume of about 0.01 mL to about 0.15 mL. In some embodiments, the handmuscle may be injected with at least about 0.01 mL of the therapeuticcomposition. In some embodiments, the hand muscle may be injected withat most about 0.15 mL of the therapeutic composition. In someembodiments, the hand muscle may be injected with greater than 0.01 mL,greater than 0.02 mL, greater than 0.03 mL, greater than 0.04 mL,greater than 0.05 mL, greater than 0.06 mL, greater than 0.07 mL,greater than 0.08 mL, greater than 0.09 mL, greater than 0.10 mL,greater than 0.11 mL, greater than 0.12 mL, greater than 0.13 mL, orgreater than 0.14 mL of the therapeutic composition.

In some aspects, the effectiveness of an administration of a therapeuticcomposition of the disclosure may be determined by conducting testsbefore administration, after administration, or both. In some cases, thetest may measure a hand function before administration, afteradministration, or both. In some cases, the test may be a tip pinchstrength test, a Moberg pickup test, or both. In some cases, the testmay be a patient-centric overall quality of life measure such as theCanadian Occupational Performance Measure (COPM).

In some aspects, the dose of a therapeutic composition of the disclosuremay be adjusted after determining the effectiveness of a prioradministration. In some cases, the dose of the PGE2 compound, themyotoxin, or both, may be increased. In other cases, the dose of thePGE2 compound, the myotoxin, or both, may be decreased. In some cases,the dose of the PGE2 compound, the myotoxin, or both, may not bechanged.

In some embodiments, the frequency of administration of a therapeuticcomposition of the disclosure (e.g., comprising a PGE2 compound and/or amyotoxin) may be adjusted after determining the effectiveness of anadministration of the therapeutic composition. In some cases, only asingle administration of the therapeutic composition may be needed totreat impaired thumb function. In some cases, two, three, four, five, ormore than five administrations of the therapeutic composition may beneeded to treat impaired thumb function. In some cases, the frequency ofadministration may be increased after determining the effectiveness of aprior administration. In other cases, the frequency of administrationmay be decreased after determining the effectiveness of a prioradministration.

In some embodiments, a subject having, suspected of having, or at riskof having impaired thumb function may be identified by a test prior toadministration of a therapeutic composition of the disclosure. In somecases, the test may be a tip pinch strength test, a Moberg pickup test,or both.

In some aspects, methods are provided for the treatment of muscleimpairment caused by compression peripheral nerve injuries. In somecases, the methods comprise administering a therapeutic composition ofthe disclosure (e.g., comprising a PGE2 compound and/or a myotoxin) to asubject having, suspected of having, or at risk of developing acompression peripheral nerve injury. For example, cubital tunnelsyndrome results from ulnar nerve entrapment at the elbow and is thesecond most common entrapment of the upper limb. Ulnar nerve compressionresults in pain or paraesthesia in the fourth and fifth finger. Inaddition to the numbness and tingling in the hand and fingers, muscleatrophy is a common result of ulnar nerve compression. In anotherexample, thoracic outlet syndrome results from compression of the nervesbetween the shoulder and neck, known as the brachial plexus. This cancause pain, weakness, numbness, tingling, a cold sensation, or sometimesa more general type of discomfort in one or both upper limbs. The commonsymptoms are pain, numbness, and tingling that radiates below theshoulder down towards the hand and usually into the pinky and ringfinger. This condition also can lead to muscle atrophy of the intrinsicmuscles of the hand.

Plantar Fasciitis

The plantar fascia is the thick tissue on the bottom of the foot. Itconnects the heel bone to the toes and creates the arch of the foot.When this tissue becomes swollen or inflamed, it is called plantarfasciitis. Plantar fasciitis is one of the most common causes of heelpain and has been estimated to affect about two million people in theUS, resulting in more than one million visits to both primary carephysicians and foot specialists. Plantar fasciitis affects bothsedentary and athletic people and is thought to result from chronicoverload either from lifestyle or exercise. Current literature suggeststhat plantar fasciitis is more correctly termed fasciosis because of thechronicity of the disease and the evidence of degeneration rather thaninflammation.

Muscle weakness may be a potential cause of chronic plantar fasciitis.Studies have shown that in a load-bearing limb, there are both passiveand active mechanisms that support the medial longitudinal arch. Supportis achieved passively by a tensioned plantar fascia, and actively byparticipation of the plantar intrinsic foot muscles (PIFM) and tibialisposterior (TP) muscle. Plantar fasciitis has been associated with PIFMatrophy at the forefoot. The forefoot volume of a foot with chronicplantar fasciitis was on average 5.2% less than the contralateralhealthy foot. The forefoot muscles include flexor hallucis brevismedialis, flexor hallucis brevis lateralis, adductor hallucistransverse, adductor hallucis oblique and the plantar interossei.Patients with abductor digiti minimi atrophy also have a significantlygreater frequency of Achilles tendinosis, calcaneal edema, calcanealspur, plantar fasciitis, and posterior tibialis tendon dysfunction thanthose without. Additionally, strengthening a weakened or atrophiedflexor digitorum brevis could also help stabilize the mediallongitudinal arch.

In some aspects, methods are provided for the treatment of impaired footfunction. In some cases, the methods comprise administering atherapeutic composition of the disclosure (e.g., comprising a PGE2compound and/or a myotoxin) to a subject having, suspected of having, orat risk of developing impaired foot function. In some embodiments, theimpaired foot function is due to plantar fasciitis. Accordingly, in someembodiments, the therapeutic composition may be suitable to treatplantar fasciitis. In some embodiments, the therapeutic composition maytreat the impaired foot function by inducing muscle generation in a footmuscle of a subject in need thereof. In some cases, the foot muscle is aplantar intrinsic foot muscle, flexor hallucis medialis, flexor hallucisbrevis lateralis, adductor hallucis transverse, adductor hallucisoblique, dorsal and plantar interossei, abductor digiti minimi, andflexor digitorum brevis.

In some embodiments, a therapeutic composition of the disclosure may beadministered in combination with foot surgery. In some cases, thetherapeutic composition can be administered before surgery, duringsurgery, after surgery, or any combination thereof. In other cases, thetherapeutic composition may be administered without foot surgery.

In some embodiments, the therapeutic composition may be administered incombination with another treatment. In some cases, the another treatmentcomprises a nonsteroidal anti-inflammatory medication, stretching, anight splint, custom orthotics, a corticosteroid injection, aplatelet-rich plasma injection, extracorporeal shock wave therapy,fasciotomy, or a combination thereof.

In some embodiments, a therapeutic composition of the disclosure may beadministered by topical administration, intradermal administration,intramuscular administration, or a combination. In some cases, thetherapeutic composition may be administered by intramuscularadministration. In some cases, the intramuscular administration maycomprise injection of a foot muscle. In some cases, the foot muscle maycomprise plantar intrinsic foot muscle, flexor hallucis medialis, flexorhallucis brevis lateralis, adductor hallucis transverse, adductorhallucis oblique, dorsal and plantar interossei, abductor digiti minimi,and flexor digitorum brevis.

In some cases, an anesthetic may be administered to a foot to a subjectin need thereof prior to injection of a therapeutic composition of thedisclosure. In some cases, the foot muscle may be injected with surgicalexposure; in other cases, the foot muscle may be injected withoutsurgical exposure. In some embodiments, a 20-, 21-, 22-, 23-, 24- or25-gauge needle may be used to inject the foot muscle.

In some embodiments, methods of treating impaired foot function mayinclude administering (e.g., intramuscular injection) a therapeuticcomposition of the disclosure to a foot muscle of a subject in needthereof, in a volume of about 0.01 mL to about 0.15 mL. In someembodiments, the foot muscle may be injected with at least about 0.01 mLof the therapeutic composition. In some embodiments, the foot muscle maybe injected with at most about 0.15 mL of the therapeutic composition.In some embodiments, the foot muscle may be injected with greater than0.01 mL, greater than 0.02 mL, greater than 0.03 mL, greater than 0.04mL, greater than 0.05 mL, greater than 0.06 mL, greater than 0.07 mL,greater than 0.08 mL, greater than 0.09 mL, greater than 0.10 mL,greater than 0.11 mL, greater than 0.12 mL, greater than 0.13 mL, orgreater than 0.14 mL of the therapeutic composition.

In some aspects, the effectiveness of an administration of a therapeuticcomposition of the disclosure may be determined by conducting testsbefore administration, after administration, or both. In some cases, thetests may measure a foot function before administration, afteradministration, or both. In some cases, the test may be a physical examto determine pain on the bottom of a foot, pain along the sole of afoot, flat feet, high arches, foot swelling, foot redness, stiffness ortightness of the arch in the bottom of a foot, or a combination thereof.

In some embodiments, the dose of the therapeutic composition may beadjusted after determining the effectiveness of a prior administration.In some cases, the dose of the PGE2 compound, the myotoxin, or both, maybe increased. In other cases, the dose of the PGE2 compound, themyotoxin, or both, may be decreased. In some cases, the dose of the PGE2compound, the myotoxin, or both may not be changed.

In some embodiments, the frequency of administration of a therapeuticcomposition of the disclosure to treat impaired foot may be adjustedafter determining the effectiveness of an administration of thetherapeutic composition. In some cases, only a single administration ofthe therapeutic composition may be needed to treat impaired footfunction. In some cases, two, three, four, five, or more than fiveadministrations of the therapeutic composition may be needed to treatimpaired foot function. In some cases, the frequency of administrationis increased after determining the effectiveness of a prioradministration. In other cases, the frequency of administration isdecreased after determining the effectiveness of a prior administration.

In some embodiments, a subject in need of treatment for impaired footfunction may be identified by a test prior to administration of thetherapeutic composition. In some cases, the test may be a physical examto determine pain on the bottom of a foot, pain along the sole of afoot, flat feet, high arches, foot swelling, foot redness, stiffness ortightness of the arch in the bottom of a foot, or a combination thereof.

Foot Drop

Foot drop, also known as drop foot, is a term for difficulty lifting thefront part of the foot. In some case, the front of the foot may drag onthe ground during walking. Foot drop impairs ambulation and can resultin falls. Foot drop is caused by weakness or paralysis of one or more ofthe muscles involved in lifting the front part of the foot. There areseveral causes of foot drop. For example, compression of a nerve in aleg that controls the muscles involved in lifting the foot, the peronealnerve, is a common cause of foot drop. This nerve may also be injuredduring hip or knee replacement surgery. A nerve root injury, “pinchednerve”, in the spine can also cause foot drop. Diabetes may also make asubject more susceptible to nerve disorders, which are associated withfoot drop. Various forms of muscular dystrophy that cause progressivemuscle weakness. In addition, disorders that affect the spinal cord orbrain, such as amyotrophic lateral sclerosis (ALS), multiple sclerosisor stroke, may cause foot drop.

Described herein are methods of treating foot drop comprisingadministering a therapeutic composition comprising a PGE2 compound and amyotoxin to a subject in need thereof. In some embodiments, thetherapeutic composition treats the impaired foot function by inducingmuscle generation in a foot or lower leg muscle of a subject in needthereof. In some cases, the foot or lower leg muscle is the anteriortibialis muscle, fibularis tertius, extensor digitorum longus, extensorhallucis longus, or a combination thereof.

In some embodiments, the therapeutic composition is administered incombination with surgery (e.g., nerve surgery). In those embodiments,the therapeutic composition can be administered before surgery, duringsurgery, after surgery, or any combination thereof. In otherembodiments, the therapeutic composition is administered without foot orlower leg surgery.

In some embodiments, the therapeutic composition is administered incombination with another treatment. In some cases, the another treatmentcomprises braces or splints, physical therapy, nerve stimulation, nervesurgery, or a combination thereof.

In some embodiments, the therapeutic composition comprising a PGE2compound and a myotoxin is administered by topical administration,intradermal administration, intramuscular administration, or acombination. In some cases, the therapeutic composition is administeredby intramuscular administration. In some cases, the intramuscularadministration comprises injection of a foot or lower leg muscle. Thefoot or lower leg muscle may comprise the anterior tibialis muscle,fibularis tertius, extensor digitorum longus, extensor hallucis longus.In some cases, an anesthetic may be administered to a foot or lower legof a subject in need prior to injection. In some cases, the foot orlower leg muscle is injected with surgical exposure; in other cases, thefoot or lower leg muscle is injected without surgical exposure. In someembodiments, a 20-, 21-, 22-, 23-, 24- or 25-gauge needle is used toinject the foot or lower leg muscle.

In some embodiments, a foot or lower leg muscle of a subject in need isinjected with a volume of about 0.01 mL to about 0.15 mL of therapeuticcomposition comprising a PGE2 compound and a myotoxin. In someembodiments, the foot or lower leg muscle is injected with at leastabout 0.01 mL of therapeutic composition. In some embodiments, the footor lower leg muscle is injected with at most about 0.15 mL oftherapeutic composition. In some embodiments, the foot or lower legmuscle is injected with greater than 0.01 mL, greater than 0.02 mL,greater than 0.03 mL, greater than 0.04 mL, greater than 0.05 mL,greater than 0.06 mL, greater than 0.07 mL, greater than 0.08 mL,greater than 0.09 mL, greater than 0.10 mL, greater than 0.11 mL,greater than 0.12 mL, greater than 0.13 mL, or greater than 0.14 mL oftherapeutic composition comprising a PGE2 compound and a myotoxin.

The effectiveness of an administration of the therapeutic compositionmay be determined by conducting tests before administration, afteradministration, or a combination thereof. The tests may measure a footor lower leg function before and/or after administration. The test maybe a physical exam to observe gait, to determine weakness of legmuscles, to determine numbness of shin, foot, and/or toes, or acombination thereof.

In some embodiments, the dose of the therapeutic composition may beadjusted after determining the effectiveness of a prior administration.In some cases, the dose of the PGE2 compound and/or the myotoxin mayincrease. In other cases, the dose of the PGE2 compound and/or themyotoxin may decrease. In some cases, the dose of the PGE2 compoundand/or the myotoxin may stay the same.

In some embodiments, the frequency of administration of the therapeuticcomposition comprising a PGE2 compound and/or the myotoxin may beadjusted after determining the effectiveness of an administration of thetherapeutic composition. In some cases, a single administration of thetherapeutic composition is needed to treat impaired foot or lower legfunction. In some cases, two or more, three or more, four or more, fiveor more administrations of the therapeutic composition are needed. Insome cases, the frequency of administration is increased afterdetermining the effectiveness of a prior administration. In other cases,the frequency of administration is decreased after determining theeffectiveness of a prior administration.

Diabetic Neuropathy

Diabetes is the most common cause of neuropathy in US and neuropathiesare the most common complication of diabetes mellitus. Atrophy of thesmall muscles of the foot is common in diabetes and is related toperipheral motor neuropathy. In long-term diabetic patients, muscleweakness and atrophy in the lower leg muscles, including the footmuscles, is common in neuropathic patients compared to non-neuropathicpatients. Volume of the intrinsic foot muscles has been shown to belower in neuropathic diabetic patients compared to non-neuropathicdiabetic patients and non-diabetic controls. Atrophy of the intrinsicmuscles of the foot can lead to fixed claw and hammer toe deformities,which are common in neuropathic diabetic patients. Additionally, theintrinsic muscles of the foot stabilize the arches of the foot. Higharches are common with the atrophy of plantar intrinsic muscles likeAbductor Hallucis, Flexor Hallucis Brevis and Adductor Hallucis.

In some aspects, methods are provided for the treatment of diabeticneuropathy or associated disorders. In some cases, the methods maycomprise administering a therapeutic composition of the disclosure(e.g., comprising a PGE2 compound and/or a myotoxin) to a subjecthaving, suspected of having, or at risk of developing a diabeticneuropathy or an associated disorder. In some cases, the disorderassociated with a diabetic neuropathy may include, without limitation,atrophy of the small muscles of the foot, muscle weakness and atrophy ofthe lower leg muscles, and atrophy of the intrinsic muscles of the foot.In some cases, the methods comprise administering the therapeuticcomposition to the small muscles of the foot, to the lower leg muscles,or to the intrinsic muscles of the foot.

Disuse-Induced Muscle Atrophy

Loss of skeletal muscle mass occurs frequently in clinical settingsfollowing limb immobilization, bed rest, spinal cord injury andpartial/complete peripheral nerve damage, resulting in significant lossof muscle mass and force production. The extent of muscle atrophy underdisuse conditions is variable and can be dependent on a variety offactors including age, the physiological function and fiber typecomposition of the muscle, and the degree of unloading and inactivity.Disuse-induced atrophy will likely affect every person in his or herlifetime, and can be debilitating especially in the elderly. Currently,there are no good pharmacological strategies to treat disuse-inducedmuscle atrophy.

Described herein are methods of treating disuse-induced muscle atrophycomprising administering a therapeutic composition comprising a PGE2compound and a myotoxin to a subject in need thereof. In someembodiments, the affected muscle has experienced unloading, inactivity,or a combination, for greater than 1 day, greater than 5 days, greaterthan 10 days, greater than 50 days, or greater than 100 days. In someembodiments, the therapeutic composition treats disuse-induced atrophyby inducing muscle generation in a muscle of a subject in need thereof,wherein the muscle has experienced unloading and/or inactivity of aprolonged period of time. In some cases, the muscle is a skeletalmuscle. In some embodiments, the therapeutic composition comprising aPGE2 compound and a myotoxin is administered by intramuscularadministration. In some cases, the intramuscular administrationcomprises injection of the affected muscle.

In some embodiments, disuse-induced muscle atrophy is due to a distalradius fracture, also known as Colles fracture. A Colles fractureresults in a backward and outward position of the hand in relation tothe wrist. It is common in the elderly. Loss of muscle mass can occurdue to immobilization. Described herein are methods of treating muscleatrophy due to a distal radius fracture comprising administering atherapeutic composition comprising a PGE2 compound and a myotoxin to asubject in need thereof. In some embodiments, the therapeuticcomposition treats the muscle atrophy by inducing muscle generation in amuscle of a subject in need thereof. In some cases, the muscle is theflexor carpi radialis, flexor pollicis longus, flexor digitorumsuperficialis, flexor digitorum profundus, flexor carpi ulnaris,extensor carpi radialis brevis/longus, extensor pollicis longus,extensor digitiorum communis, extensor carpi ulnaris or a combinationthereof. In some embodiments, the therapeutic composition isadministered in combination with surgery (e.g., wrist arthroscopy). Inthose embodiments, the therapeutic composition can be administeredbefore surgery, during surgery, after surgery, or any combinationthereof. In other embodiments, the therapeutic composition isadministered without surgery.

In some embodiments, the therapeutic composition is administered incombination with another treatment. In some cases, the another treatmentcomprises braces or splints, physical therapy, nerve stimulation, nervesurgery, or a combination thereof.

In some embodiments, the therapeutic composition comprising a PGE2compound and a myotoxin is administered by topical administration,intradermal administration, intramuscular administration, or acombination. In some cases, the therapeutic composition is administeredby intramuscular administration. In some cases, the intramuscularadministration comprises injection of a hand or lower arm muscle. Thehand or lower arm muscle may comprise the flexor carpi radialis, flexorpollicis longus, flexor digitorum superficialis, flexor digitorumprofundus, flexor carpi ulnaris, extensor carpi radialis brevis/longus,extensor pollicis longus, extensor digitiorum communis, extensor carpiulnaris, or a combination thereof.

In some cases, an anesthetic may be administered to a hand or lower armof a subject in need prior to injection. In some cases, the hand orlower arm muscle is injected with surgical exposure; in other cases, thehand or lower arm muscle is injected without surgical exposure. In someembodiments, a 20-, 21-, 22-, 23-, 24- or 25-gauge needle is used toinject the hand or lower arm leg muscle.

In some embodiments, a hand or lower arm muscle of a subject in need isinjected with a volume of about 0.01 mL to about 0.15 mL of therapeuticcomposition comprising a PGE2 compound and a myotoxin. In someembodiments, the hand or lower arm muscle is injected with at leastabout 0.01 mL of therapeutic composition. In some embodiments, the handor lower arm muscle is injected with at most about 0.15 mL oftherapeutic composition. In some embodiments, the hand or lower armmuscle is injected with greater than 0.01 mL, greater than 0.02 mL,greater than 0.03 mL, greater than 0.04 mL, greater than 0.05 mL,greater than 0.06 mL, greater than 0.07 mL, greater than 0.08 mL,greater than 0.09 mL, greater than 0.10 mL, greater than 0.11 mL,greater than 0.12 mL, greater than 0.13 mL, or greater than 0.14 mL oftherapeutic composition comprising a PGE2 compound and a myotoxin.

The effectiveness of an administration of the therapeutic compositionmay be determined by conducting tests before administration, afteradministration, or a combination thereof. The tests may measure a handor lower arm function before and/or after administration. The test mayinclude range of motion of wrist, grip strength, patient reportedoutcomes such as the Disability of Arm Shoulder and Hand or acombination thereof.

In some embodiments, the dose of the therapeutic composition may beadjusted after determining the effectiveness of a prior administration.In some cases, the dose of the PGE2 compound and/or the myotoxin mayincrease. In other cases, the dose of the PGE2 compound and/or themyotoxin may decrease. In some cases, the dose of the PGE2 compoundand/or the myotoxin may stay the same.

In some embodiments, the frequency of administration of the therapeuticcomposition comprising a PGE2 compound and/or the myotoxin may beadjusted after determining the effectiveness of an administration of thetherapeutic composition. In some cases, a single administration of thetherapeutic composition is needed to treat impaired hand or lower armfunction. In some cases, two or more, three or more, four or more, fiveor more administrations of the therapeutic composition are needed. Insome cases, the frequency of administration is increased afterdetermining the effectiveness of a prior administration. In other cases,the frequency of administration is decreased after determining theeffectiveness of a prior administration.

In some embodiments, disuse-induced muscle atrophy is due to a hipfracture. A hip fracture may be due to major or minor trauma. In elderlypeople with bones weakened by osteoporosis, relatively little trauma,even walking, may result in a hip fracture. Loss of muscle mass canoccur due to joint immobilization, and bed rest. Loss of muscle can beinduced by a combination of unloading and inactivity. Weakness of thehip muscle is an often-occurring condition after displaced fractures ofthe proximal femur in older patients. Described herein are methods oftreating muscle atrophy due to a hip fracture comprising administering atherapeutic composition comprising a PGE2 compound and a myotoxin to asubject in need thereof. In some embodiments, the therapeuticcomposition treats the muscle atrophy by inducing muscle generation in ahip muscle of a subject in need thereof. In some cases, the hip muscleis iliacus, psoas major, gluteus maximus, gluteus medius, gluteusminimus, tensor fasciae latae, superior gemellus, inferior gemellus,obturator internus, obturator externus, quadratus femoris, piriformis,adductor magnus, adductor longus, adductor brevis, adductor minimus,pectineus, rectus femoris, vastus lateralis, vastus medialis, vastusintermedius, quadriceps femoris, Sartorius, biceps femoris,semitendinosus, semimembranosus, psoas minor, iliopsoas, gracilis, or acombination thereof.

In some embodiments, the therapeutic composition is administered incombination with surgery (e.g., joint arthroplasty). In thoseembodiments, the therapeutic composition can be administered beforesurgery, during surgery, after surgery, or any combination thereof. Inother embodiments, the therapeutic composition is administered withoutsurgery.

In some embodiments, the therapeutic composition is administered incombination with another treatment regimen. In some cases, the anothertreatment regimen comprises braces or splints, physical therapy, nervestimulation, nerve surgery, or a combination thereof.

In some embodiments, the therapeutic composition comprising a PGE2compound and a myotoxin is administered by topical administration,intradermal administration, intramuscular administration, or acombination. In some cases, the therapeutic composition is administeredby intramuscular administration. In some cases, the intramuscularadministration comprises injection of a hip muscle. In some cases, thehip muscle is iliacus, psoas major, gluteus maximus, gluteus medius,gluteus minimus, tensor fasciae latae, superior gemellus, inferiorgemellus, obturator internus, obturator externus, quadratus femoris,piriformis, adductor magnus, adductor longus, adductor brevis, adductorminimus, pectineus, rectus femoris, vastus lateralis, vastus medialis,vastus intermedius, quadriceps femoris, Sartorius, biceps femoris,semitendinosus, semimembranosus, psoas minor, iliopsoas, gracilis, or acombination thereof.

In some cases, an anesthetic may be administered to a hip of a subjectin need prior to injection. In some cases, the hip muscle is injectedwith surgical exposure; in other cases, the hip muscle is injectedwithout surgical exposure. In some embodiments, a 20-, 21-, 22-, 23-,24- or 25-gauge needle is used to inject the hip muscle.

In some embodiments, a hip muscle of a subject in need is injected witha volume of about 0.5 mL to about 5 mL of therapeutic compositioncomprising a PGE2 compound and a myotoxin. In some embodiments, the hipmuscle is injected with at least about 0.5 mL of therapeuticcomposition. In some embodiments, the hip muscle is injected with atmost about 4 mL of therapeutic composition. In some embodiments, the hipmuscle is injected with greater than 0.5 mL, greater than 1.0 mL,greater than 1.5 mL, greater than 2.0 mL, greater than 2.5 mL, greaterthan 3.0 mL, greater than 3.5 mL of therapeutic composition comprising aPGE2 compound and a myotoxin.

The effectiveness of an administration of the therapeutic compositionmay be determined by conducting tests before administration, afteradministration, or a combination thereof. The tests may measure ahip-related function before and/or after administration. The test may bestrength testing of hip and knee muscles as well as functional testssuch as the get up and go test or a combination thereof.

In some embodiments, the dose of the therapeutic composition may beadjusted after determining the effectiveness of a prior administration.In some cases, the dose of the PGE2 compound and/or the myotoxin mayincrease. In other cases, the dose of the PGE2 compound and/or themyotoxin may decrease. In some cases, the dose of the PGE2 compoundand/or the myotoxin may stay the same.

In some embodiments, the frequency of administration of the therapeuticcomposition comprising a PGE2 compound and/or the myotoxin may beadjusted after determining the effectiveness of an administration of thetherapeutic composition. In some cases, a single administration of thetherapeutic composition is needed to treat impaired hand or lower armfunction. In some cases, two or more, three or more, four or more, fiveor more administrations of the therapeutic composition are needed. Insome cases, the frequency of administration is increased afterdetermining the effectiveness of a prior administration. In other cases,the frequency of administration is decreased after determining theeffectiveness of a prior administration.

In some embodiments, disuse-induced muscle atrophy is due to a rotatorcuff injury. In some cases, a rotator cuff injury may be due to an acuterotator cuff tear. In some cases, a rotator cuff injury may be due todegenerative and/or chronic rotator cuff tear. A degenerative and/orchronic rotator cuff tear may be due to repetitive stress, lack of bloodsupply, bone spurs, or a combination thereof. People over 40 years oldare at a greater risk for a rotator cuff tear. Loss of muscle mass canoccur due to lack of tension on muscle as well as joint stiffness. Lossof muscle can be induced by a combination of unloading and inactivity.Described herein are methods of treating muscle atrophy due to a rotatorcuff injury comprising administering a therapeutic compositioncomprising a PGE2 compound and a myotoxin to a subject in need thereof.In some embodiments, the therapeutic composition treats the muscleatrophy by inducing muscle generation in a rotator cuff muscle of asubject in need thereof. In some cases, the rotator cuff muscle issupraspinatus, infraspinatus, subscapularis, teres minor, or acombination thereof.

In some embodiments, the therapeutic composition is administered incombination with surgery (e.g., rotator cuff arthroscopy). In thoseembodiments, the therapeutic composition can be administered beforesurgery, during surgery, after surgery, or any combination thereof. Inother embodiments, the therapeutic composition is administered withoutsurgery.

In some embodiments, the therapeutic composition is administered incombination with another treatment regimen. In some cases, the anothertreatment regimen comprises braces or splints, physical therapy, nervestimulation, nerve surgery, or a combination thereof.

In some embodiments, the therapeutic composition comprising a PGE2compound and a myotoxin is administered by topical administration,intradermal administration, intramuscular administration, or acombination. In some cases, the therapeutic composition is administeredby intramuscular administration. In some cases, the intramuscularadministration comprises injection of a rotator cuff muscle. In somecases, the rotator cuff muscle is supraspinatus, infraspinatus,subscapularis, teres minor, or a combination thereof.

In some cases, an anesthetic may be administered to a rotator cuffmuscle of a subject in need prior to injection. In some cases, therotator cuff muscle is injected with surgical exposure; in other cases,the rotator cuff muscle is injected without surgical exposure. In someembodiments, a 20-, 21-, 22-, 23-, 24- or 25-gauge needle is used toinject the rotator cuff muscle.

In some embodiments, a rotator cuff muscle of a subject in need isinjected with a volume of about 0.5 mL to about 5 mL of therapeuticcomposition comprising a PGE2 compound and a myotoxin. In someembodiments, the rotator cuff muscle is injected with at least about 0.5mL of therapeutic composition. In some embodiments, the rotator cuffmuscle is injected with at most about 4 mL of therapeutic composition.In some embodiments, the rotator cuff muscle is injected with greaterthan 0.5 mL, greater than 1.0 mL, greater than 1.5 mL, greater than 2.0mL, greater than 2.5 mL, greater than 3.0 mL, greater than 3.5 mL oftherapeutic composition comprising a PGE2 compound and a myotoxin.

The effectiveness of an administration of the therapeutic compositionmay be determined by conducting tests before administration, afteradministration, or a combination thereof. The tests may measure arotator cuff-related function before and/or after administration. Thetest may be strength testing of shoulder abduction, external rotationand forward flexion, full and empty can test, drop arm test, and TheQuality of Life Outcome Measure for Rotator Cuff or a combinationthereof.

In some embodiments, the dose of the therapeutic composition may beadjusted after determining the effectiveness of a prior administration.In some cases, the dose of the PGE2 compound and/or the myotoxin mayincrease. In other cases, the dose of the PGE2 compound and/or themyotoxin may decrease. In some cases, the dose of the PGE2 compoundand/or the myotoxin may stay the same.

In some embodiments, the frequency of administration of the therapeuticcomposition comprising a PGE2 compound and/or the myotoxin may beadjusted after determining the effectiveness of an administration of thetherapeutic composition. In some cases, a single administration of thetherapeutic composition is needed to treat impaired hand or lower armfunction. In some cases, two or more, three or more, four or more, fiveor more administrations of the therapeutic composition are needed. Insome cases, the frequency of administration is increased afterdetermining the effectiveness of a prior administration. In other cases,the frequency of administration is decreased after determining theeffectiveness of a prior administration.

E. Methods of Treating Pelvic Floor Disorders

Pelvic floor disorders (PFDs) arise from dysfunction of the pelvic floormuscles. The pelvic floor muscles are often damaged by childbirth andpelvic surgery. Pelvic floor disorders also arise from other trauma,aging, obesity, neurological diseases, and other injuries. The mostcommon pelvic floor disorders are due, at least in part, to decreasedfunction of the pelvic floor muscles. These disorders include stressurinary incontinence, overactive bladder/urinary urgency incontinence,mixed urinary incontinence, pelvic organ prolapse, and fecalincontinence.

Pelvic floor disorders comprise a wide variety of conditions in both menand women, although women are more commonly affected. The pelvic floorprovides anatomic support for the pelvic organs (bladder, prostate,rectum, uterus vagina) and is integral to the proper function of theurinary system, to sexual and reproductive function, and to colorectalfunction. The pelvic floor is comprised of the pelvic floor muscles aswell as the relevant connective tissue (ligaments, tendons, andoverlying fascia). The pelvic floor muscles include the levator ani andthe coccygeus. The levator ani has three parts: the pubococcygeus, theiliococcygeus and the puborectalis.

Strengthening or improving function of the pelvic floor muscles maytreat these types of pelvic floor disorders. In addition, strengtheningor improving function of the pelvic floor muscles may prevent pelvicfloor disorders from developing in patients identified as high risk(e.g., after complicated delivery, after certain types of pelvicsurgery). This application may also be used in combination with existingtreatments for pelvic floor disorders (e.g., muscletraining/biofeedback, neuromodulation, pharmacotherapy, surgery) inorder to improve treatment outcomes.

Provided herein are methods of treating or preventing pelvic floordisorders. In some cases, the methods may comprise administering atherapeutic composition of the disclosure (e.g., comprising a PGE2compound and/or a myotoxin) to a subject having, suspected of having, orat risk of developing a pelvic floor disorder. In some cases, the pelvicfloor disorder is selected from the group consisting of stress urinaryincontinence, overactive bladder/urinary urgency incontinence, mixedurinary incontinence, pelvic organ prolapse, and fecal incontinence. Insome aspects, the methods may comprise administering a therapeuticcomposition of the disclosure to the pelvic floor muscles of a subjectin need thereof. In some cases, the pelvic floor muscles comprise thelevator ani, the coccygeus, or both. In some cases, the levator aniincludes the pubococcygeus, the iliococcygeus, and the puborectalis.

In one aspect, provided herein are applications of a therapeuticcomposition comprising a prostaglandin E2 (PGE2) compound and amyotoxin, as described elsewhere herein, the genitourinary system. Insome embodiments, the therapeutic composition can be administered toimprove a function of the genitourinary system. In further embodiments,the therapeutic composition can be administered to enhance theeffectiveness of an existing approach to treat a disease or disorder ofthe genitourinary system. The disease or disorder of the genitourinarysystem can be a urological disorder, gynecological disorder, acolorectal disorder, or a combination thereof.

The pelvic floor muscles support the bladder and the urethra. Mostadults can hold over 400 mL of urine in the bladder. Urine flows fromthe bladder through the urethra to the outside. Around the opening ofthe bladder is the sphincter muscle. It squeezes to prevent urine fromleaking through the urethra. Impairment of any of these muscles canresult a urological disorder such as stress incontinence. Moreover,strengthening of any of these muscles can be targeted to treaturological disorders such overactive bladder disorder and underactivebladder disorder.

Stress Urinary Incontinence

Stress urinary incontinence occurs when the bladder leaks urine duringphysical activity or exertion such as coughing, lifting of heavyobjects, exercise, or change in positions. It occurs when any of themuscles that control the ability to hold urine in the bladder is weak orimpaired in function. When any one of the muscles, such as the skeletalexternal urethral muscle, the pubic urethral muscle, becomes weak, urinecan pass when pressure is place on the bladder. Weakened muscles may becaused by childbirth, injury to the urethra area, medications, surgeryin the pelvic area such as prostate surgery, progressive atrophy anddiminished contractility of the skeletal muscles, nerve damage. Currenttreatments include surgical interventions and injection of “bulkingagents”.

Described herein are methods of treating stress urinary incontinence(SUI) comprising administering a therapeutic composition comprising aPGE2 compound and a myotoxin to a subject in need thereof. In someembodiments, the therapeutic composition treats SUI by inducing muscleregeneration a muscle of a subject in need thereof. In some cases, themuscle is the skeletal external urethral muscle, the pubic urethralmuscle, or the external urethral sphincter muscle.

In some embodiments, the therapeutic composition is administered incombination with surgery (e.g., prostate surgery). In those embodiments,the therapeutic composition can be administered before surgery, duringsurgery, after surgery, or any combination thereof. In otherembodiments, the therapeutic composition is administered withoutsurgery.

In some embodiments, the therapeutic composition comprising a PGE2compound and a myotoxin is administered by intramuscular administration.In some cases the intramuscular administration comprises injection ofthe skeletal external urethral muscle, the pubic urethral muscle, andthe external urethral sphincter muscle. In some cases, an anesthetic maybe administered prior to injection. In some cases, the muscle isinjected with surgical exposure; in other cases, the muscle is injectedwithout surgical exposure. In some embodiments, an 18-, 19-, 20-, 21-,22- or 23-gauge needle is used to inject the muscle.

In some embodiments, a muscle of a subject in need is injected with avolume of about 2 mL or less of therapeutic composition comprising aPGE2 compound and a myotoxin. In some embodiments, the muscle isinjected with at least about 2 mL of therapeutic composition. In someembodiments, the muscle is injected with less than 2 mL, less than 1.8mL, less than 1.6 mL, less than 1.4 mL, less than 1.2 mL, less than 1mL, less than 0.8 mL, less than 0.6 mL, less than 0.4 mL or less than0.2 mL of therapeutic composition comprising a PGE2 compound and amyotoxin.

The effectiveness of an administration of the therapeutic compositionmay be determined by conducting tests before administration, afteradministration, or a combination thereof. The test may be cystoscopy, apad weight tests, a voiding diary, pelvic or abdominal ultrasound,post-void residual (PVR), urinalysis, urinary stress test, urodynamicstudies such as leak point pressure, x-rays with contrast dye, or acombination thereof. In some embodiments, the dose of the therapeuticcomposition may be adjusted after determining the effectiveness of aprior administration. In some cases, the dose of the PGE2 compoundand/or the myotoxin may increase. In other cases, the dose of the PGE2compound and/or the myotoxin may decrease. In some cases, the dose ofthe PGE2 compound and/or the myotoxin may stay the same. In someembodiments, the frequency of administration of the therapeuticcomposition comprising a PGE2 compound and/or the myotoxin may beadjusted after determining the effectiveness of an administration of thetherapeutic composition. In some cases, a single administration of thetherapeutic composition is needed. In some cases, two or more, three ormore, four or more, five or more administrations of the therapeuticcomposition are needed. In some cases, the frequency of administrationis increased after determining the effectiveness of a prioradministration. In other cases, the frequency of administration isdecreased after determining the effectiveness of a prior administration.

Mixed Incontinence or Overactive bladder

Overactive bladder (OAB) is a condition in which the bladder squeezesurine out at the wrong time. For example, a person suffering from anoveractive bladder may urinate eight or more times a day or two or moretimes at night; may have a sudden and strong need to urinateimmediately; or leak urine after a sudden, strong urge to urinate.Several conditions may contribute to signs and symptoms of overactivebladder including neurological disorders (e.g., stroke, multiplesclerosis), diabetes, diuretics, urinary tract infections, tumors,bladder stones, enlarged prostate, constipation, excess consumption ofcaffeine or alcohol, declining cognitive function due to aging, andincomplete bladder emptying. Strengthening and improving function of theexternal urethral sphincter muscle can impact positive and negativereflex actions between the bladder and the urethra. For example,improved contractile action of the urethral sphincter muscle can signalthe bladder to inhibit flow of urine.

Described herein are methods of treating overactive bladder (OAB)comprising administering a therapeutic composition comprising a PGE2compound and a myotoxin to a subject in need thereof. In someembodiments, the therapeutic composition treats OAB by inducing muscleregeneration a muscle of a subject in need thereof. In some cases, themuscle is the skeletal external urethral muscle.

In some embodiments, the therapeutic composition is administered incombination with surgery (e.g., prostate surgery). In those embodiments,the therapeutic composition can be administered before surgery, duringsurgery, after surgery, or any combination thereof. In otherembodiments, the therapeutic composition is administered withoutsurgery.

In some embodiments, the therapeutic composition comprising a PGE2compound and a myotoxin is administered by intramuscular administration.In some cases the intramuscular administration comprises injection ofthe skeletal external urethral muscle. In some cases, an anesthetic maybe administered prior to injection. In some cases, the muscle isinjected with surgical exposure; in other cases, the muscle is injectedwithout surgical exposure. In some embodiments, an 18-, 19-, 20-, 21-,22- or 23-gauge needle is used to inject the muscle.

In some embodiments, a muscle of a subject in need is injected with avolume of about 2 mL or less of therapeutic composition comprising aPGE2 compound and a myotoxin. In some embodiments, the muscle isinjected with at least about 2 mL of therapeutic composition. In someembodiments, the muscle is injected with less than 2 mL, less than 1.8mL, less than 1.6 mL, less than 1.4 mL, less than 1.2 mL, less than 1mL, less than 0.8 mL, less than 0.6 mL, less than 0.4 mL or less than0.2 mL of therapeutic composition comprising a PGE2 compound and amyotoxin.

The effectiveness of an administration of the therapeutic compositionmay be determined by conducting tests before administration, afteradministration, or a combination thereof. The test may be post-voidresidual (PVR) test, urine flow rate test, bladder pressure test, or acombination thereof. In some embodiments, the dose of the therapeuticcomposition may be adjusted after determining the effectiveness of aprior administration. In some cases, the dose of the PGE2 compoundand/or the myotoxin may increase. In other cases, the dose of the PGE2compound and/or the myotoxin may decrease. In some cases, the dose ofthe PGE2 compound and/or the myotoxin may stay the same. In someembodiments, the frequency of administration of the therapeuticcomposition comprising a PGE2 compound and/or the myotoxin may beadjusted after determining the effectiveness of an administration of thetherapeutic composition. In some cases, a single administration of thetherapeutic composition is needed. In some cases, two or more, three ormore, four or more, five or more administrations of the therapeuticcomposition are needed. In some cases, the frequency of administrationis increased after determining the effectiveness of a prioradministration. In other cases, the frequency of administration isdecreased after determining the effectiveness of a prior administration.

Obstetrical anal Sphincter Injury (OASIS)

Obstetrical anal sphincter injury (OASIS) is damage to the analsphincter during childbirth, and can lead to significant comorbidities,including anal incontinence, rectovaginal fistula and pain. They aremore commonly associated with force deliveries than vacuum-assistedvaginal deliveries. They are also associated with an increased risk ofpostpartum urinary retention. Surgical repair of the anal sphincter maynot completely restore muscle function.

Described herein are methods of treating obstetrical anal sphincterinjury comprising administering a therapeutic composition comprising aPGE2 compound and a myotoxin to a subject in need thereof. In someembodiments, the therapeutic composition treats OAB by inducing muscleregeneration of an anal muscle of a subject in need thereof. In somecases, the anal muscle is the external anal sphincter muscle, or theinner anal sphincter muscle.

In some embodiments, the therapeutic composition is administered incombination with surgery (e.g., external anal sphincter repair, internalanal sphincter repair). In those embodiments, the therapeuticcomposition can be administered before surgery, during surgery, aftersurgery, or any combination thereof. In other embodiments, thetherapeutic composition is administered without surgery.

In some embodiments, the therapeutic composition comprising a PGE2compound and a myotoxin is administered by intramuscular administration.In some cases, the intramuscular administration comprises injection ofthe skeletal external anal sphincter muscle. In some cases, ananesthetic may be administered prior to injection. In some cases, themuscle is injected with surgical exposure; in other cases, the muscle isinjected without surgical exposure. In some embodiments, an 18-, 19-,20-, 21-, 22- or 23-gauge needle is used to inject the muscle.

In some embodiments, an anal sphincter muscle of a subject in need isinjected with a volume of about 2 mL or less of therapeutic compositioncomprising a PGE2 compound and a myotoxin. In some embodiments, themuscle is injected with at least about 2 mL of therapeutic composition.In some embodiments, the muscle is injected with less than 2 mL, lessthan 1.8 mL, less than 1.6 mL, less than 1.4 mL, less than 1.2 mL, lessthan 1 mL, less than 0.8 mL, less than 0.6 mL, less than 0.4 mL or lessthan 0.2 mL of therapeutic composition comprising a PGE2 compound and amyotoxin.

The effectiveness of an administration of the therapeutic compositionmay be determined by conducting tests before administration, afteradministration, or a combination thereof. In some embodiments, the doseof the therapeutic composition may be adjusted after determining theeffectiveness of a prior administration. In some cases, the dose of thePGE2 compound and/or the myotoxin may increase. In other cases, the doseof the PGE2 compound and/or the myotoxin may decrease. In some cases,the dose of the PGE2 compound and/or the myotoxin may stay the same. Insome embodiments, the frequency of administration of the therapeuticcomposition comprising a PGE2 compound and/or the myotoxin may beadjusted after determining the effectiveness of an administration of thetherapeutic composition. In some cases, a single administration of thetherapeutic composition is needed. In some cases, two or more, three ormore, four or more, five or more administrations of the therapeuticcomposition are needed. In some cases, the frequency of administrationis increased after determining the effectiveness of a prioradministration. In other cases, the frequency of administration isdecreased after determining the effectiveness of a prior administration.

Fecal Incontinence

Fecal incontinence, also known as bowel incontinence or encopresis, isthe inability to control bowel movement, causing stool/feces to leakunexpectedly from the rectum. It ranges from an occasional leakage ofstool while passing gas, to a complete loss of bowel control. Causesinclude diarrhea, constipation, muscle damage or never damage. In somecases, muscle damage is due to childbirth, or is associated with aging.

Described herein are methods of treating fecal incontinence comprisingadministering a therapeutic composition comprising a PGE2 compound and amyotoxin to a subject in need thereof. In some embodiments, thetherapeutic composition treats fetal incontinence by inducing muscleregeneration of an anal muscle of a subject in need thereof. In somecases, the anal muscle is the external anal sphincter muscle, or theinner anal sphincter muscle.

In some embodiments, the therapeutic composition is administered incombination with surgery (e.g., external anal sphincter repair, internalanal sphincter repair). In those embodiments, the therapeuticcomposition can be administered before surgery, during surgery, aftersurgery, or any combination thereof. In other embodiments, thetherapeutic composition is administered without surgery.

In some embodiments, the therapeutic composition comprising a PGE2compound and a myotoxin is administered by intramuscular administration.In some cases, the intramuscular administration comprises injection ofthe skeletal external anal sphincter muscle. In some cases, ananesthetic may be administered prior to injection. In some cases, themuscle is injected with surgical exposure; in other cases, the muscle isinjected without surgical exposure. In some embodiments, an 18-, 19-,20-, 21-, 22- or 23-gauge needle is used to inject the muscle.

In some embodiments, an anal sphincter muscle of a subject in need isinjected with a volume of about 2 mL or less of therapeutic compositioncomprising a PGE2 compound and a myotoxin. In some embodiments, themuscle is injected with at least about 2 mL of therapeutic composition.In some embodiments, the muscle is injected with less than 2 mL, lessthan 1.8 mL, less than 1.6 mL, less than 1.4 mL, less than 1.2 mL, lessthan 1 mL, less than 0.8 mL, less than 0.6 mL, less than 0.4 mL or lessthan 0.2 mL of therapeutic composition comprising a PGE2 compound and amyotoxin.

The effectiveness of an administration of the therapeutic compositionmay be determined by conducting tests before administration, afteradministration, or a combination thereof. In some embodiments, the doseof the therapeutic composition may be adjusted after determining theeffectiveness of a prior administration. In some cases, the dose of thePGE2 compound and/or the myotoxin may increase. In other cases, the doseof the PGE2 compound and/or the myotoxin may decrease. In some cases,the dose of the PGE2 compound and/or the myotoxin may stay the same. Insome embodiments, the frequency of administration of the therapeuticcomposition comprising a PGE2 compound and/or the myotoxin may beadjusted after determining the effectiveness of an administration of thetherapeutic composition. In some cases, a single administration of thetherapeutic composition is needed. In some cases, two or more, three ormore, four or more, five or more administrations of the therapeuticcomposition are needed. In some cases, the frequency of administrationis increased after determining the effectiveness of a prioradministration. In other cases, the frequency of administration isdecreased after determining the effectiveness of a prior administration.

F. Methods of Treating Gastroesophageal Reflux Disease

With a prevalence of 10-20% in the adult population, gastroesophagealreflux disease (GERD) is one of the most common diseases of the uppergastreointestinal tract. GERD occurs when the ring of muscles at thebottom of the esophagus is weakened or damaged, allowing gastric acid toenter the distal esophagus. The acid stimulates the chemoreceptors,causing irritation and leads to the onset of symptoms. Esophagealsymptoms (e.g., heartburn) and extraesophageal symptoms (e.g., oral,pharyngeal, laryngeal, and pulmonary disorders) of GERD are triggered bymucosal exposure to the gastric acid, and are related to the frequencyof reflux events and the duration of mucosal acidification. Other GERDsymptoms may include epigastric fullness, pressure or pain, dyspepsia,nausea, bloating, belching, chronic cough, bronchospasm, wheezing,hoarseness, and asthma.

The antireflux barrier includes two sphincters—the lower esophagealsphincter (LES) and the diaphragmatic sphincter at the gastroesophagealjunction. The two sphincters maintain tonic closure and augmented reflexclosure of the sphincter mechanism. The LES is composed of smoothmuscles, and it maintains tonic contraction owing to myogenic as well asneurogenic factors. The diaphragmatic sphincter is composed of striatedmuscles that also exhibit tone and contracts due to the excitatorynerves. The mammalian diaphragm is primarily a respiratory muscle.However, it consists of two separate muscles: the crural and the costaldiaphragms. The costal diaphragm is a respiratory muscle, while thecrural diaphragm has two functions: respiratory and gastrointestinal.The crural diaphragm is composed of skeletal muscle. Contraction of thediaphragmatic sphincter provides a powerful sphincter mechanism at thelower end of the esophagus contributing to both tonic (sustained) andphasic pressure increases at the level of the LES. A crural myotomystudy has demonstrated that there was a significant increase inspontaneous acid reflux. After removal of the crural diaphragm,intrinsic esophageal muscle cannot fully compensate for the loss of thecrural muscle. In humans, the diaphragmatic hiatus is the site ofminimum GEJ opening aperture, and hiatal hernia has shown excess refluxindicating that the crural diaphragm has a crucial barrier role. A studyhas also shown that patients with esophagitis may have a thinner cruraldiaphragm and a deficient GEJ activity during forced inhalation. Theanatomical changes and functional failure of the crural diaphragm isesophagitis patients supports the possibility of a skeletal muscledeficiency in GERD.

The primary treatment of GERD is acid suppression which can be achievedwith several classes of mechanisms including antacids,histamine-receptor antagonists or proton-pump inhibitors. Surgicaltherapy may include laparoscopic fundoplication or bariatric surgery.Complications from anti-reflux surgery may include dysphagia ofsufficient severity to require esophageal dilation in about 6% ofpatients treated with fundoplication surgery as well as a significantincrease in flatulence and the inability to belch (gas bloat syndrome).

In some aspects, methods are provided for treating gastroesophagealreflux disease (GERD). In some cases, the methods may compriseadministering a therapeutic composition of the disclosure (e.g., a PGE2compound and/or a myotoxin) to a subject having, suspected of having, orat risk of developing GERD. In some embodiments, the therapeuticcomposition may treat GERD by inducing muscle regeneration in the cruraldiaphragm.

In some embodiments, a therapeutic composition of the disclosure may beadministered in combination with a surgical procedure to treat GERD. Insome cases, the therapeutic composition can be administered beforesurgery, during surgery, after surgery, or any combination thereof. Insome cases, the crural diaphragm may be accessed laparoscopically andinjected with a therapeutic composition of the disclosure. In somecases, the location of the crural diaphragm may be identified bylocating the esophagus and the diaphragm hiatus, and then injecting thediaphragm adjacent to the esophagus with a therapeutic compositionprovided herein. In some cases, the injections may be performedcircumferentially around the esophagus. In some cases, at least one,two, three, four, five, six, seven, eight, nine, ten, or more than tencircumferential injections around the esophagus may be performed. Insome embodiments, a 27-, 28-, 29- or 30-gauge needle may be used toinject the crural diaphragm.

In some embodiments, methods of treating GERD may include administering(e.g., intramuscular injection) a therapeutic composition of thedisclosure to the crural diaphragm of a subject in need thereof, in avolume of about 0.05 mL to about 0.15 mL. In some embodiments, thecrural diaphragm may be injected with at least about 0.05 mL of thetherapeutic composition. In some embodiments, the crural diaphragm maybe injected with at most about 0.15 mL of the therapeutic composition.In some embodiments, the crural diaphragm may be injected with greaterthan 0.05 mL, greater than 0.06 mL, greater than 0.07 mL, greater than0.08 mL, greater than 0.09 mL, greater than 0.10 mL, greater than 0.11mL, greater than 0.12 mL, greater than 0.13 mL, or greater than 0.14 mLof the therapeutic composition.

In some aspects, the effectiveness of an administration of a therapeuticcomposition of the disclosure (e.g., to treat GERD) may be determined byconducting tests before administration, after administration, or both.In some cases, the test may include the Heartburn Specific Quality ofLife (HBQQL) questionnaire, the GERD Health-Related Quality of Life(GERD-HRQL) questionnaire, or both. In some cases, the test may includemonitoring an increase in thickness of the crural diaphragm usingendoscopic ultrasound.

G. Methods of Treating Obstructive Sleep Apnea

Obstructive sleep apnea (OSA), apnea, or hypopnea, is characterized byrepetitive episodes of complete or partial obstructions of the upperairway during sleep. The upper airway is divided into three regions: thenasopharynx, the oropharynx, and the hypopharynx. The oropharynx issubdivided into the retropalatal region (the posterior margin of thehard palate to the caudal margin of the soft palate) and theretroglossal region (the caudal margin of the soft palate to the base ofthe epiglottis). The majority of patients with OSA have upper airwaynarrowing in the retropalatal region, the retroglossal region, or both.

The skeletal muscles surrounding the pharyngeal airway are phasicallyactivated during inspiration, which may help to dilate the airway andstiffen the airway walls. The pharyngeal muscles help regulate theposition of the soft palate, tongue, hyoid apparatus, and posterolateralpharyngeal walls. Contraction of specific muscles within the palatalmuscles opens the airway in the retropalatal region. Pharyngeal musclescan have different effects when activated in concert as opposed to whenactivated individually. Coactivation of the muscles in the anteriorpharyngeal wall such as the geniohyoid and sternohyoid act on the hyoidbone to move it ventrally. The tensor palatine moves the soft palateventrally. The genioglossus acts to displace the tongue ventrally. Theextrinsic muscles of the tongue consist of the genioglossus, hyoglossus,and syloglossus and are important for the protrusion and retraction ofthe tongue. The genioglossus is the primary protruder muscle of thetongue with its contraction playing a seminal role in keeping thepharyngeal airway open, mainly by widening the oropharynx in theanterior-poasterior dimension. During respiration, the primary goal ofthe pharyngeal muscles is to keep the airway open allowing for the flowof air in and out of the lung.

In healthy individuals, the pharyngeal muscles are able to adequatelycompensate for the increase in airway resistance to maintain a patentairway. However, individuals that have a narrow upper airway, either dueto obesity or bony structures crowding the airway, are at an increasedrisk of pharyngeal collapse during sleep. OSA is characterized byincreased collapsibility of the upper airway during sleep, which resultsin reduced airflow (hypopnea) or blocked airflow (apnea) resulting inintermittent hypoxia. The upper airway may collapse because dilatormuscles may be unable to sustain patency during portions of therespiratory cycle. The genioglossus is the major upper airway dilatormuscle. In OSA patients, the genioglossus muscles have been shown to bestructurally and functionally abnormal. One potential mechanism by whichthese upper airway dilating muscles fail is by fatigue. Fatigue is theloss in the muscle capacity for developing force resulting from muscleactivity under load, and which is reversible by rest. In OSA, the upperairway dilating muscles are subjected to repeated bursts of forcefulcontraction at the end of each obstructive apnea, which may occurseveral hundred times each night. The frequency and duration ofobstructive apneas are greater in the latter part of the night in OSApotentially implicating genioglossus fatigue as a contributing factor.

Given the importance of upper airway muscles in maintaining patency,decreased muscle function could contribute to pharyngeal closure. Inorder to adapt to increased contractile demands, skeletal muscle fiberphenotype can under a shift from oxidative slow-twitch, fatigueresistant Type I, to glycolytic fast-twitch Type II fibers, whichgenerate increased force but are more prone to fatigue. Fast-twitchmuscle fibers fatigue more rapidly than slow-twitch fibers. An increasein fast-twitch fibers would be expected to increase the fatigueabilityof the upper airway muscles, leaving the airway susceptible to collapseand leading to a cycle of increasingly severe episodes as the level offatigue increases following repeated activation during the night.Although the genioglossus is only one of many muscles that act inconcert to prevent flow limitation in the pharynx, it may substantiallyimprove pharyngeal patency when activated adequately to obtain optimalanterior displacement of the tongue.

The reduction in tension and strength of the upper airway muscles may beone of the key factors in the etiology of OSA. These muscles may includethe genioglossus and the tensor palatine. In some cases, improving thestrength of these muscles may improve the patency of the upper airway,and reducing the symptoms of OSA. In addition, other upper airwaymuscles that may contribute to upper airway patency may include thegeniohyoid muscles. Targeting these muscles, either individually or incombination, may improve upper airway patency to treat OSA. Although anumber of factors may contribute to OSA, upper airway collapsibility andanatomy is fundamentally important in OSA pathogenesis. Accordingly, OSApatients with upper airway pharyngeal muscle weakness may be potentialtargets for this therapy.

Continuous positive airway pressure (CPAP) is the standard treatment formoderate-to-severe OSA. However, the nasal mask required for CPAP duringsleep leads to poor acceptance and compliance rates. Oral appliance (OA)therapy is also widely used for the treatment of moderate and severeOSA. These consist of a maxillary and mandibular splint which hold thelower jaw forward during sleep. However, the efficacy of OA is inferiorto CPAP. Upper airway stimulation augments neural drive of thepharyngeal muscle by unilaterally stimulating the hypoglossal nerve. Themain limitations of this approach are the risky surgical procedure toimplant the device, a high upfront cost and variable response. Manysurgical approaches have been proposed like RF ablation of the tonguebase, genioglossus advancement, hyoid suspension, maxillomandibularadvancement, and tongue base suspension. As with any surgicalintervention, these approaches carry the risk of surgical complicationsand high upfront cost. Furthermore, drug therapies based on a number ofmechanisms have been proposed with limited success. These include anincrease in tone in the upper airway dilator muscles, an increase inventilatory drive, a reduction in the proportion of rapid eye movement(REM) sleep, an increase in cholinergic tone during sleep, an increasein arousal threshold, a reduction in airway resistance, and a reductionin surface tension in the upper airway.

OSA has various pathophysiologic causes including an anatomicallycompromised or collapsible upper airway, inadequate responsiveness ofthe upper airway dilator muscles during sleep (minimal increase in EMGactivity to negative pharyngeal pressure), waking up prematurely toairway narrowing (a low respiratory arousal threshold), or having anoversensitive ventilatory control system. Hence, effective treatmentsmay require treating a primary cause of OSA or treating a combination ofcauses.

In some aspects, methods are provided for treating obstructive sleepapnea (OSA). In some cases, the methods may comprise administering atherapeutic composition of the disclosure (e.g., comprising a PGE2compound and/or a myotoxin) to a subject having, suspected of having, orat risk of developing OSA. In some cases, the methods may compriseadministering a therapeutic composition of the disclosure to a subjecthaving, suspected of having, or at risk of developing hypopnea. In somecases, the methods may comprise administering a therapeutic compositionof the disclosure to a subject having, suspected of having, or at riskof developing apnea. In some cases, the methods may compriseadministering a therapeutic composition of the disclosure to a subjectto complement or enhance the efficacy of an additional therapy orintervention to treat OSA.

In some embodiments, the therapeutic composition may treat obstructivesleep apnea, hypopnea, apnea, or a combination thereof, by inducingmuscle regeneration in a muscle of the upper airway of a subject. Insome cases, the upper airway muscle comprises the genioglossus. In somecases, the upper airway muscle comprises the tensor palatine. In somecases, the upper airway muscle comprises the geniohyoid muscles. In somecases, OSA, hypopnea, or apnea can be treated by administering atherapeutic composition of the disclosure (e.g., comprising a PGE2compound and/or a myotoxin) to the upper airway muscle (e.g., thegenioglossus, the tensor palatine, the geniohyoid muscles, or anycombination thereof).

In some embodiments, methods of treating OSA, hypopnea, or apnea mayinclude administering a therapeutic composition of the disclosure (e.g.,comprising a PGE2 compound and/or a myotoxin) to a subject in needthereof by topical administration, intradermal administration,intramuscular administration, or a combination thereof. In some cases,the therapeutic composition may be administered by intramuscularadministration. In some cases, the intramuscular administration maycomprise injection of an upper airway muscle. The upper airway musclemay include the genioglossus, the tensor palatine, the geniohyoidmuscles, or a combination thereof. In some cases, an anesthetic may beadministered to the upper airway prior to injection of the therapeuticcomposition. In some cases, the upper airway muscle may be injected withsurgical exposure; in other cases, the upper airway muscle may beinjected without surgical exposure. In some cases, surgical proceduresto alter the anatomy of the upper airway may be enhanced by increasingthe strength of upper airway muscles (e.g., by administering atherapeutic composition of the disclosure to the upper airway muscles).In some embodiments, a 27-, 28-, 29- or 30-gauge needle may be used toinject the upper airway muscle.

In some embodiments, methods of treating OSA, hypopnea, apnea, or acombination thereof may include administering (e.g., intramuscularinjection) a therapeutic composition of the disclosure to an upperairway muscle of a subject in need thereof, in a volume of about 0.01 mLto about 0.15 mL. In some embodiments, the upper airway muscle may beinjected with at least about 0.01 mL of a therapeutic composition of thedisclosure. In some embodiments, the upper airway muscle may be injectedwith at most about 0.15 mL of a therapeutic composition of thedisclosure. In some embodiments, the upper airway muscle may be injectedwith greater than 0.01 mL, greater than 0.02 mL, greater than 0.03 mL,greater than 0.04 mL, greater than 0.05 mL, greater than 0.06 mL,greater than 0.07 mL, greater than 0.08 mL, greater than 0.09 mL,greater than 0.10 mL, greater than 0.11 mL, greater than 0.12 mL,greater than 0.13 mL, or greater than 0.14 mL of a therapeuticcomposition of the disclosure.

In some aspects, the effectiveness of an administration of a therapeuticcomposition may be determined by conducting tests before administration,after administration, or both. In some cases, the test may assessclinical benefits for an OSA patient using the apnea/hypopnea index(AHI) and the level of daytime sleepiness associated with OSA, estimatedby the Epworth Sleepiness Scale (ESS). In some cases, the test mayinclude polysomnographic parameters (e.g., AI, HI, RERArl, Arl, LSat),Sleep Related Quality of Life (FOSQ), and Reaction Time Testing (PVT).

In some embodiments, a dose of the therapeutic composition may beadjusted after determining the effectiveness of a prior administration.In some cases, the dose of the PGE2 compound, the myotoxin, or both, maybe increased. In other cases, the dose of the PGE2 compound, themyotoxin, or both may be decreased. In some cases, the dose of the PGE2compound, the myotoxin, or both may not be changed. In some embodiments,the frequency of administration of a therapeutic composition of thedisclosure (e.g., comprising a PGE2 compound and/or a myotoxin) may beadjusted after determining the effectiveness of a prior administrationof the therapeutic composition. In some cases, only a singleadministration of the therapeutic composition may be needed to treatOSA, hypopnea, or apnea. In some cases, two, three, four, five, or morethan five administrations of the therapeutic composition may be neededto treat OSA, hypopnea, or apnea. In some cases, the frequency ofadministration may be increased after determining the effectiveness of aprior administration. In other cases, the frequency of administrationmay be decreased after determining the effectiveness of a prioradministration.

H. Kits

In yet another aspect of the present invention, provided herein is a kitfor promoting muscle regeneration and/or increasing muscle mass in asubject in need thereof, or for preventing or treating a musclecondition in a subject in need thereof. In some embodiments, the kitcomprises a composition described herein that comprises a combination ofa PGE2 compound (e.g., PGE2 receptor agonist) and a myotoxin. In otherembodiments, the kit comprises a pharmaceutical composition describedherein. The kit typically contains containers which may be formed from avariety of materials such as glass or plastic, and can include forexample, bottles, vials, syringes, and test tubes. A label typicallyaccompanies the kit, and includes any writing or recorded material,which may be electronic or computer readable form providing instructionsor other information for use of the kit contents.

Kits of the present invention may be suitable for treating any number ofmuscle conditions, including but not limited to muscle conditions thatare associated with muscle damage, injury, or atrophy. The kits may alsobe useful for promoting muscle regeneration in a subject in need thereofand/or increasing muscle mass. Non-limiting examples of suitableconditions for prevention or treatment with kits of the presentinvention include traumatic injury (e.g., acute muscle trauma, acutenerve trauma), acute muscle injury, acute nerve injury, chronic nerveinjury, soft tissue hand injury, carpal tunnel syndrome (CTS), Duchennemuscular dystrophy (DMD), Becker muscular dystrophy, limb girdlemuscular dystrophy, amyotrophic lateral sclerosis (ALS), distal musculardystrophy (DD), inherited myopathies, myotonic muscular dystrophy (MDD),mitochondrial myopathies, myotubular myopathy (MM), myasthenia gravis(MG), congestive heart failure, periodic paralysis, polymyositis,rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiaccachexia, stress induced urinary incontinence, sarcopenia, spinalmuscular atrophy, fecal sphincter dysfunction, Bell's palsy, rotatorcuff injury, spinal cord injury, hip replacement, knee replacement,wrist fracture, diabetic neuropathy, gastroesophageal reflux disease(GERD), obstructive sleep apnea (OSA), pelvic floor disorders (e.g.,stress urinary incontinence, overactive bladder/urinary urgencyincontinence, mixed urinary incontinence, pelvic organ prolapse, fecalincontinence), musculoskeletal disorders (e.g., impaired hand function,impaired thumb function, impaired foot function), plantar fasciitis,foot drop, disuse-induced muscle atrophy, impaired eyelid function(e.g., eyelid drooping, impaired blinking, entropion, ectropion),strabismus, nystagmus, presbyopia. Additional examples of suitableconditions for prevention or treatment with kits of the presentinvention may include muscle disorders that affect small isolatedmuscles that can be regenerated with localized transplantation of smallnumbers of cells, including: atrophy and muscle dysfunction in the faceor hand after nerve injury or direct trauma that does not recover afterreinnervation; extraocular muscle injury causing inability to move theeye and dipoplia seen in Graves' disease; traumatic injury; progressiveexternal ophthalmoplegia; and urinary and fecal incontinence.

In some embodiments, the kit further comprises isolated muscle cells. Inother embodiments, the kit further comprises instructions for use (e.g.,to the kit user). In some embodiments, the kit further comprises one ormore reagents and/or one or more devices (e.g., a delivery device) thatare used, for example, to administer a composition and/or pharmaceuticalcomposition of the present invention, to administer isolated musclecells (e.g., to a subject in need thereof), or a combination thereof.

IV. EXAMPLES

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes only, and are not intended to limit the invention in anymanner.

Example 1 Acute Prostaglandin E2 Delivery Augments Skeletal MuscleRegeneration and Strength in Aged Mice

This example illustrates that PGE2 signaling is required for muscle stemcell function during regeneration.

The elderly suffer from progressive skeletal muscle wasting andregenerative failure that decreases mobility and quality of life.Crucial to muscle regeneration are adult muscle stem cells (MuSCs) thatreside in niches in muscle tissues, poised to respond to damage andrepair skeletal muscles throughout life. During aging, the proportion offunctional MuSCs markedly decreases, hindering muscle regeneration. Todate, no therapeutic agents are in clinical use that target MuSCs tocombat this regenerative decline. Here, we identify a naturalimmunomodulator, prostaglandin E2 (PGE2), as a potent regulator of MuSCfunction essential to muscle regeneration. We found that the PGE2receptor, EP4, is essential for MuSC proliferation in vitro andengraftment in vivo in mice. In MuSCs of aged mice, the PGE2 pathway isdysregulated due to a cell intrinsic molecular defect, elevatedprostaglandin degrading enzyme (15-PGDH) that renders PGE2 inactive.This defect is overcome by transient acute exposure of MuSCs to a stabledegradation-resistant PGE2, 16,16-dimethyl PGE2 (dmPGE2), concomitantwith MuSC transplantation into injured muscles. Notably, a singleintramuscular injection of dmPGE2 alone suffices to accelerateregeneration, evident by an early increase in endogenous MuSC numbersand myofiber sizes following injury. Furthermore, aged mouse muscleforce generating capacity was increased in response to exercise-inducedregeneration and an acute dmPGE2 treatment regimen. Our findings reveala novel therapeutic indication for PGE2 as a potent inducer of muscleregeneration and strength.

To counter the decline in muscle regenerative potential we soughttherapeutic agents that target MuSCs, also known as satellite cells, astem cell population dedicated to muscle regeneration. Since a transientinflammatory and fibroadipogenic response plays a crucial role in muscleregeneration, we sought to identify inflammatory modulators induced byinjury that could overcome the age-related decline in MuSC function. Ananalysis of our transcriptome database revealed that the Ptger4 receptorfor PGE2, a natural and potent lipid mediator during acute inflammation,was expressed at high levels on freshly isolated MuSCs. In muscle tissuelysates, we detected a surge in levels of PGE2 three days after injuryto young (2-4 mo) mouse muscles by standard injury paradigms entailingnotexin injection or cryoinjury (FIGS. 2A and 6A), and a concomitantupregulation of its synthesizing enzymes, Ptges and Ptges2 (FIG. 2B).This early and transient time window coincides with the well-documentedkinetics of MuSC expansion and inflammatory cytokine accumulation postinjury. To determine if PGE2 treatment enhanced MuSC behavior, weFACS-purified MuSCs from hindlimb muscles from young mice (2-4 mo) andplated them on hydrogels of 12 kpa stiffness to maintain stem cellfunction. We found that PGE2 (10 ng/ml) increased cell division assayedby EDU incorporation (FIGS. 2B-2D) and that an acute 1-day exposure toPGE2 induced a 6-fold increase in the number of MuSCs relative tocontrols one week later (FIG. 2C).

PGE2 is known to signal through four G-protein coupled receptors (Ptger1-4; EP1-4), but the expression of these receptors in MuSCs has notpreviously been described. An analysis of the transcript levels of thedifferent receptors (Ptger1-4) revealed that the only receptorsupregulated after PGE2 treatment of MuSCs are Ptger1 and Ptger4 (FIG.6E). PGE2 stimulated MuSCs had elevated intracellular cAMP confirmingthat PGE2 signals through EP4 to promote proliferation and a stem celltranscriptional state (FIGS. 6F-6H). In the presence of an EP4antagonist, ONO-AE3-208, proliferation induced by PGE2 was blunted (FIG.2D). However, the specificity of PGE2 for EP4 was most clearly shown inMuSCs lacking the receptor following cre-mediated conditional ablation(FIGS. 2E-2G and 6I-6J). Indeed, even in the presence of growthfactor-rich media, these EP4-null MuSCs failed to proliferate. Finally,we found that MuSCs growth arrested by exposure to medium with charcoalstripped serum, divided upon addition of PGE2 (FIGS. 2H and 6K). Thus,PGE2/EP4 stands out as necessary and sufficient for MuSC proliferation.

We sought to determine if PGE2 could ameliorate the muscle regenerativedefects previously reported for aged MuSCs. By contrast with young mousemuscles (2-4 mo), notexin damage to aged muscles (18-20 mo) did not leadto an increase in PGE2 synthesis. Instead, steady state PGE2 levels inaged muscle remained unchanged post injury (FIG. 3A) and weresignificantly higher than in young limb tibialis anterior (TA) muscles(FIG. 3B). We hypothesized that the PGE2 in aged muscle might bedysfunctional due to a catabolic defect. Indeed, when we analyzed thePGE2 present in young and aged TA muscle tissues by mass spectrometry,we found that the relative amount of the inactive form,13,14-dihydro-15-keto PGE2 (PGEM), was significantly increased in theaged (FIGS. 3C-3D and 7A-7C). This proved to be due to a concomitant7-fold increase in levels of mRNA encoding the PGE2 degrading enzyme(15-PGDH), the initial step in the conversion of PGE2 to its inactiveform (FIG. 3E). In contrast, the relative levels of the prostaglandintransporter (PGT), PGE2 synthesizing enzymes, and EP4 receptor did notdiffer between young and aged MuSCs (FIGS. 8A-8C). Additionally, whenaged MuSCs were exposed to a 1-day pulse of PGE2 or to an inhibitor of15-PGDH (SW033291), the effects of 15-PGDH were overcome and thecharacteristic increase in proliferation and maintenance of Pax7expression was observed (FIGS. 3F and 8D). Like young, aged MuSCs failedto proliferate in medium comprised of charcoal stripped serum, but wererescued by addition of PGE2 alone (FIG. 3G). We surmised that in agedMuSCs the PGE2 pathway is dysregulated due to a cell intrinsic moleculardefect, elevated 15-PGDH that can be surmounted in culture by acuteexposure to PGE2 or SW (FIG. 3H).

Since aged MuSCs are heterogeneous, we sought to determine the effect ofPGE2 at the single cell level. Clonal analysis can reveal differencesthat are masked by analysis of the population as a whole. Accordingly,we performed long-term time-lapse microscopy in hydrogel ‘microwells’ ofsingle aged MuSCs transiently exposed to PGE2 for 1 day and untreatedcontrol MuSCs. Data were collected over a 48 h time period and thenanalyzed using our previously described Baxter Algorithms for CellTracking and Lineage Reconstruction. We observed a remarkable increasein cumulative cell numbers in response to PGE2, spanning 6 generationsfor the most robust clones (FIGS. 3I-3J). The numbers of cells per clonefollowing PGE2 treatment were significantly augmented due to a markedincrease in proliferation (FIGS. 3I-3J and 8E-8F) that was accompaniedby a profound reduction in cell death (FIGS. 3J and 8E-8G). Thesesynergistic effects led to the observed increases in aged MuSC numbersin response to PGE2.

To test whether transient treatment of young MuSCs with PGE2 augmentsregeneration, we transplanted cultured PGE2 treated MuSCs into injuredhindlimb muscles of mice. To monitor the dynamics of regeneration overtime in a quantitative manner in vivo, we capitalized on a sensitive andquantitative bioluminescence imaging (BLI) assay we previously developedfor monitoring MuSC function post-transplantation. MuSCs were isolatedfrom young transgenic mice (2-4 mo) expressing GFP and luciferase(GFP/Luc mice), exposed to an acute 1-day PGE2 treatment, harvested andtransplanted on day 7. Equivalent numbers of dmPGE2 treated and controlMuSCs (250 cells) were transplanted into injured hindlimbs of young (2-4mo) NOD-SCID mice. Following acute treatment with PGE2, young MuSCregenerative capacity was enhanced by an order of magnitude whenassessed by BLI (FIG. 4A). In contrast, following transplantation of4-fold greater numbers of cultured MuSCs that lacked the EP4 receptordue to conditional ablation (FIG. 4B), the BLI signal that was initiallydetected progressively declined to levels below the threshold ofsignificance (FIG. 4B).

Furthermore, when notexin injury was performed in the mouse model ofmuscle stem cell specific deletion of EP4 (Pax7^(CreERT2);EP4^(fl/fl))(FIGS. 10A-10B), muscle regeneration was impaired as observed by theelevated number of embryonic myosin heavy chain (eMHC) positive fibers(FIGS. 10C-10D). This was accompanied by the reduction incross-sectional area of the mouse fibers in thePax7^(CreERT 2);EP4^(fl/f) group, assessed at the end of theregeneration time point (day 21) (FIG. 10E). A significant reduction inforce output (tetanus) was also detected at day 14 post-injury (FIGS.10F-10G). Thus, PGE2 signaling via the EP4 receptor is required for MuSCregeneration in vivo.

To test if direct injection of PGE2 without culture could be effectivein promoting regeneration in vivo, we coinjected PGE2 together withfreshly isolated MuSCs. For all subsequent in vivo injectionexperiments, we used a modified, more stable form of PGE2,16,16-dimethyl PGE2 (dmPGE2). We hypothesized that for the aged MuSCexperiments, the delivery of the modified 15-PGDH-resistant dmPGE2 wasparticularly important, as 15-PGDH is significantly elevated in agedMuSCs (FIG. 3E). Using dmPGE2, we observed significantly enhancedengraftment of young and aged MuSCs relative to controls that wasfurther increased in response to notexin injury, a well-acceptedstringent test of stem cell function (FIGS. 4C-4D). Thus, the deliveryof dmPGE2 together with MuSC cell populations suffices to augmentregeneration.

We postulated that delivery of PGE2 alone could stimulate muscleregeneration. To test this, muscles of young mice were injured withcardiotoxin and three days later a bolus of dmPGE2 was injected into thehindlimb muscles of young mice. We observed an increase (60±15%) inendogenous PAX7-expressing MuSCs in the classic satellite cell nichebeneath the basal lamina and atop myofibers fourteen days post injury(FIGS. 5A-5B), whereas dmPGE2 had no effect in the absence of injury.Further, at this early time point, the distribution of myofibers shiftedtoward larger sizes, assessed as cross-sectional area using the BaxterAlgorithms for Myofiber Analysis, suggesting that regeneration isaccelerated by PGE2 (FIGS. 5C-5D and 9A-9B). In addition, we tracked theresponse to injury and dmPGE2 of endogenous MuSCs by luciferaseexpression using a transgenic mouse model, Pax7^(creERT2);Rosa26-LSL-Luc(FIG. 5E). The BLI data were in agreement with the histological data(FIGS. 5F-5G).

We tested the effects of injecting indomethacin, a nonsteroidalanti-inflammatory drug (NSAID) and an inhibitor of COX2 which reducesPGE2 synthesis, on muscle regeneration. Upon indomethacin injection intothe hindlimb muscles of the same Pax7^(creERT2);Rosa26-LSL-Luc mousemodel three days post-cardiotoxin injury, we observed a significantdecrease in luciferase activity indicative of an impairment in musclestem cell activation and regeneration (FIGS. 11A-11B). Injection ofindomethacin into cardiotoxin-injured muscles also led to a significantloss in Twitch force as compared to the control group assessed at day 14post-injury (FIG. 11C). In aged mice, we also detected a substantialincrease (24±2%) in the number of endogenous MuSCs (FIGS. 5H-5I), and aconcomitant increase in myofiber sizes (FIGS. 5J-5K) fourteen dayspost-injury after a single dmPGE2 injection. Thus, exposure solely todmPGE2 impacts the magnitude and time course of the endogenous repair.

As the ultimate test, we determined if dmPGE2 enhanced regenerationcould lead to increased muscle strength after a natural injury inducedby downhill treadmill-running. In this scenario, damage was caused by adaily 10 min run on a downhill treadmill 20 degree decline. During weekone, aged mice in the treatment group ran for 5 days in succession andwere injected daily with dmPGE2 after exercise. During week two, agedmice in the treatment group ran for 5 consecutive days but received noadditional treatment (FIG. 5L). The specific twitch and tetanic forcewere compared for dmPGE2 treated and untreated gastrocnemius mousemuscles (GA) and both were significantly increased (FIGS. 5M-5P). Thus,an acute exposure to dmPGE2 concurrent with exercise-induced injury canconfer a significant increase in aged muscle strength.

We have discovered a new indication for PGE2 in skeletal muscleregeneration. Prior studies of PGE2 effects on skeletal muscle haveshown that it alters the proliferation, fusion, protein degradation, anddifferentiation of myoblasts in tissue culture. Thus, these studiesdiffer from ours as myoblasts are progenitors that have lost stem cellfunction. Satellite cells (MuSCs) are crucial to development andregeneration and their numbers are increased by running or other highintensity exercise in young and aged mice and humans. Non-steroidalanti-inflammatory agents have been reported to attenuate theexercise-induced increase in MuSCs. Our data provide novel evidence thatthe beneficial effects of the early transient wave of inflammation thatcharacterizes efficacious muscle regeneration is due in part to PGE2 andits receptor EP4, which are essential and sufficient for MuSCproliferation and engraftment. For hematopoietic, liver, and colontissues, delivery of the inhibitor of 15-PGDH, SW033291, was recentlyshown to enhance regeneration. Notably, PGE2 and its analogues havesafely been used in human patients for decades, for instance to inducelabor and to promote hematopoietic stem cell transplantation paving theway for its clinical use in restoring muscles post-injury. In summary,our findings show that an acute PGE2 regimen suffices to rapidly androbustly enhance regeneration of exercise-induced damage and overcomeage-associated limitations leading to increased strength.

Mice: We performed all experiments and protocols in compliance with theinstitutional guidelines of Stanford University and Administrative Panelon Laboratory Animal Care (APLAC). We obtained wild-type aged C57BL/6(18-20 mo) mice from the US National Institute on Aging (NIA) for agedmuscle studies and young wild-type C57BL/6 mice from Jackson Laboratory.Double-transgenic GFP/luc mice were generated as described previously¹.Briefly, mice expressing a firefly luciferase (luc) transgene under theregulation of the ubiquitous Actb promoter were maintained in the FVBstrain. Mice expressing a green fluorescent protein (GFP) transgeneunder the regulation of the ubiquitous UBC promoter were maintained inthe C57BL/6 strain. We used cells from GFP/luc for allogenictransplantation experiments into NOD-SCID (Jackson Laboratory) recipientmice. EP4^(flox/flox) (EP4^(f/f)) mice were a kind gift from K.Andreasson (Stanford University)². Double-transgenicPax7^(CreERT 2);Rosa26-LSL-Luc were generated by crossing Pax7^(CreERT2)mice obtained from Jackson Laboratory (Stock #017763)³ andRosa26-LSL-Luc obtained from Jackson Laboratory (Stock #005125)⁴. Wevalidated these genotypes by appropriate PCR-based strategies. All micefrom transgenic strains were of young age. Young mice were 2-4 months(“mo”) of age and aged mice were 18-20 months of age for all strains.All mice used in these studies were females.

Muscle stem cell isolation: We isolated and enriched muscle stem cellsas previously described^(1,5,6). Briefly, a gentle collagenase digestionand mincing by the MACs Dissociator enabled numerous single fibers to bedissociated, followed by dispase digestion to release mononucleatedcells from their niches. Subsequently, the cell mixture was depleted forhematopoietic lineage expressing and non-muscle cells(CD45⁻/CD11b⁻/CD31⁻) using a magnetic bead column (Miltenyi). Theremaining cell mixture was then subjected to FACS analysis to sort forMuSCs co-expressing CD34 and α7-integrin markers. We generated andanalyzed flow cytometry scatter plots using FlowJo v10.0. For each sort,we pooled together MuSCs (5,000 each) from at least three independentdonor female mice.

Muscle stem cell transplantation: We transplanted 250 MuSCs (FIGS. 4A,4C, and 4D) or 1,000 MuSCs (FIG. 4B) immediately following FACSisolation or after collection from cell culture directly into thetibialis anterior (TA) muscles of recipient mice as previouslydescribed^(1,5,6). For young MuSC studies, we transplanted cells fromGFP/luc mice (2-4 mo of age) into hindlimb-irradiated NOD-SCID mice. Foraged MuSCs studies, we transplanted cells from aged C57BL/6 mice (18-20mo, NIH) that were transduced with a luc-IRES-GFP lentivirus (GFP/lucvirus) on day 2 of culture for a period of 24 hr before transplantation,as previously described⁵ (see below “Muscle stem cell culture, treatmentand lentiviral infection” section for details). Prior to transplantationof muscle stem cells, we anesthetized NOD-SCID recipient mice withketamine (2.4 mg per mouse) by intraperitoneal injection. We thenirradiated hindlimbs with a single 18 Gy dose, with the rest of the bodyshielded in a lead jig. We performed transplantations within 2 d ofirradiation.

Cultured cells were treated as indicated (vehicle or PGE2 treated 10ng/ml) and collected from hydrogel cultures by incubation with 0.5%trypsin in PBS for 2 min at 37° C. and counted using a hemocytometer. Weresuspended cells at desired cell concentrations in 0.1% gelatin/PBS andthen transplanted them (250 MuSCs per TA) by intramuscular injectioninto the TA muscles in a 10 μl volume. For fresh MuSCs transplantation,we coinjected sorted cells with 13 nmol of 16,16-Dimethyl ProstaglandinE2 (dmPGE2) (Tocris, catalog #4027) or vehicle control (PBS). Wecompared cells from different conditions by transplantation into the TAmuscles of contralateral legs in the same mice. One month aftertransplant, we injected 10 μl of notexin (10 μg ml⁻¹; Latoxan, France)to injure recipient muscles and to activate MuSCs in vivo. Eight weeksafter transplantation, mice were euthanized and the TAs were collectedfor analysis.

Bioluminescence imaging: We performed bioluminescence imaging (BLI)using a Xenogen-100 system, as previously described^(1,5,6). Briefly, weanesthetized mice using isofluorane inhalation and administered 120 μLD-luciferin (0.1 mmol kg⁻¹, reconstituted in PBS; Caliper LifeSciences)by intraperitoneal injection. We acquired BLI using a 60 s exposure atF-stop=1.0 at 5 minutes after luciferin injection. Digital images wererecorded and analyzed using Living Image software (CaliperLifeSciences). We analyzed images with a consistent region-of-interest(ROI) placed over each hindlimb to calculate a bioluminescence signal.We calculated a bioluminescence signal in radiance (p s⁻¹ cm⁻² sr⁻¹)value of 10⁴ to define an engraftment threshold. This radiance thresholdof 10⁴ is approximately equivalent to the total flux threshold in p/sreported previously. This BLI threshold corresponds to the histologicaldetection of one or more GFP+ myofibers^(1,5,6). We performed BLIimaging every week after transplantation.

Muscle injury: We used an injury model entailing intramuscular injectionof 10 μl of notexin (10 μg ml⁻¹; Latoxan) or cardiotoxin (10 μM;Latoxan) into the TA muscle. For cryoinjury, an incision was made in theskin overlying the TA muscle and a copper probe, chilled in liquidnitrogen, was applied to the TA muscle for three 10 s intervals,allowing the muscle to thaw between each application of the cryoprobe.When indicated, 48 hr after injury either 16,16-Dimethyl ProstaglandinE2 (dmPGE2) (13 nmol, Tocris, catalog #4027) or vehicle control (PBS)was injected into the TA muscle. The contralateral TA was used as aninternal control. We collected tissues 14 days post-injury for analysis.

For Pax7^(CreERT2); Rosa26-LSL-Luc mice experiments, we treated micewith five consecutive daily intraperitoneal injections of tamoxifen toactivate luciferase expression under the control of the Pax? promoter. Aweek after the last tamoxifen injection, mice were subjected tointramuscular injection of 10 μl of cardiotoxin (10 μM; Latoxan), whichwe designated as day 0 of the assay. Three days later either 13 nmoldmPGE2 (13 nmol) or vehicle control (PBS) was injected into the TAmuscle. The contralateral TA was used as an internal control.Bioluminescence was assayed at days 3, 7, 10 and 14 post-injury.

Tissue histology: We collected and prepared recipient TA muscle tissuesfor histology as previously described^(5,6). We incubated transversesections with anti-LAMININ (Millipore, clone A5, catalog #05-206,1:200), and anti-PAX7 (Santa Cruz Biotechnology, catalog #sc-81648,1:50) primary antibodies and then with AlexaFluor secondary Antibodies(Jackson ImmunoResearch Laboratories, 1:200). We counterstained nucleiwith DAPI (Invitrogen). We acquired images with an AxioPlan2epifluorescent microscope (Carl Zeiss Microimaging) with Plan NeoFluar10×/0.30NA or 20×/0.75NA objectives (Carl Zeiss) and an ORCA-ER digitalcamera (Hamamatsu Photonics) controlled by the SlideBook (3i) software.The images were cropped using Adobe Photoshop with consistent contrastadjustments across all images from the same experiment. The imagecomposites were generated using Adobe Illustrator. We analyzed thenumber of PAX7 positive cells using the MetaMorph Image Analysissoftware (Molecular Devices), and the fiber area using the BaxterAlgorithms for Myofiber Analysis that identified the fibers andsegmented the fibers in the image to analyze the area of each fiber. ForPAX7 quantification we examined serial sections spanning a depth of atleast 2 mm of the TA. For fiber area at least 10 fields ofLAMININ-stained myofiber cross-sections encompassing over 400 myofiberswere captured for each mouse as above. Data analyses were blinded. Theresearchers performing the imaging acquisition and scoring were unawareof treatment condition given to sample groups analyzed.

Hydrogel fabrication: We fabricated polyethylene glycol (PEG) hydrogelsfrom PEG precursors, synthesized as described previously⁶. Briefly, weproduced hydrogels by using the published formulation to achieve 12-kPa(Young's modulus) stiffness hydrogels in 1 mm thickness which is theoptimal condition for culturing MuSCs and maintaining stem cell fate inculture⁶. We fabricated hydrogel microwell arrays of 12-kPa for clonalproliferation experiments, as described previously⁶. We cut and adheredall hydrogels to cover the surface area of 12-well or 24-well cultureplates.

Muscle stem cell culture, treatment and lentiviral infection: Followingisolation, we resuspended MuSCs in myogenic cell culture mediumcontaining DMEM/F10 (50:50), 15% FBS, 2.5 ng ml⁻¹ fibroblast growthfactor-2 (FGF-2 also known as bFGF) and 1% penicillin-streptomycin. Weseeded MuSC suspensions at a density of 500 cells per cm² surface area.We maintained cell cultures at 37° C. in 5% CO₂ and changed mediumdaily. For PGE2, 15-PGDH inhibitor and EP4 receptor antagonist treatmentstudies, we added 1-200 ng/ml Prostaglandin E2 (Cayman Chemical) (unlessspecified in the figure legends, 10 ng/ml was the standard concentrationused), and/or 1 μM EP4 antagonist (ONO-AE3-208, Cayman Chemical), or 1μM 15-PGDH inhibitor (SW033291, Cayman Chemical) to the MuSCs culturedon collagen coated dishes for the first 24 h. The cells were thentrypsinized and cells reseeded onto hydrogels for an additional 6 daysof culture. All treatments were compared to their solvent (DMSO) vehiclecontrol. For stripped serum assays, we resuspended isolated MuSCs inmedium containing DMEM/F10 (50:50), 15% charcoal stripped FBS (Gibco,cat #12676011), 2.5 ng ml⁻¹ bFGF and 1% penicillin-streptomycin. Whennoted in the figure, we additionally added 1.5 μg/ml insulin (Sigma,10516) and 0.25 μM dexamethasone (Sigma, D8893) to stripped serum cellmedium. For these experiments MuSCs were cultured on hydrogels andvehicle (DMSO) or 10 ng/ml PGE2 (Cayman Chemical) was added to thecultures with every media change (every two days). Proliferation (seebelow) was assayed 7 days later.

We performed all MuSC culture assays and transplantations after 1 weekof culture unless noted otherwise. For aged MuSCs transplant studies, weinfected MuSCs with lentivirus encoding elongation factor-1αpromoter-driven luc-IRES-GFP (GFP/luc virus) for 24 h in culture asdescribed previously³. For EP4^(f/f) MuSCs studies, we isolated MuSCs asdescribed above (Muscle stem cell isolation), and infected all cellswith the GFP/luc virus and a subset of them was coinfected with alentivirus encoding pLM-CMV-R-Cre (mCherry/Cre virus) for 24 h inculture. pLM-CMV-R-Cre was a gift from Michel Sadelain (Addgene plasmid#27546)⁷. We transplanted aged MuSC (250 cells) or EP4^(f/f) MuSCs(1,000 cells) into young (2-4 mo) 18-gy irradiated TAs of NOD-SCIDrecipient mice. For in vitro proliferation assays, EP4^(f/f) MuSCs wereplated on hydrogels post-infection and treated for 24 hr with vehicle(DMSO) or 10 ng/ml PGE2, and proliferation was assayed 3 days later.Cells were assayed for GFP and/or mCherry expression 48 h post-infectionusing an inverted fluorescence microscope (Carl Zeiss Microimaging).MuSCs are freshly isolated from the mice by FACS and put in culture fora maximum time period of one week, therefore mycoplasma contamination isnot assessed.

Proliferation assays: To assay proliferation, we used three differentassays (hemocytometer, VisionBlue, and EdU). For each, we seeded MuSCson flat hydrogels (hemocytometer and VisionBlue) or collagen-coatedplates (EdU assay) at a density of 500 cells per cm² surface area. Forhemocytometer cell number count, we collected cells at indicatedtimepoints by incubation with 0.5% trypsin in PBS for 5 min at 37° C.and quantified them using a hemocytometer at least 3 times.Additionally, we used the VisionBlue Quick Cell Viability FluorometricAssay Kit (BioVision, catalog #K303) as a readout for cell growth inculture. Briefly, we incubated MuSCs with 10% VisionBlue in culturemedium for 3 h, and measured fluorescence intensity on a fluorescenceplate reader (Infinite M1000 PRO, Tecan) at Ex=530-570 nm, Em=590-620nm. We assayed proliferation using the Click-iT EdU Alexa Fluor 555Imaging kit (Life Technologies). Briefly, we incubated live cells withEdU (20 μM) for 1 hr prior to fixation, and stained nuclei according tothe manufacturer's guidelines together with anti-MYOGENIN (Santa Cruz,catalog #sc576, 1:250) to assay differentiation. We counterstainednuclei with DAPI (Invitrogen). We acquired images with an AxioPlan2epifluorescent microscope (Carl Zeiss Microimaging) with Plan NeoFluar10×/0.30NA or 20×/0.75NA objectives (Carl Zeiss) and an ORCA-ER digitalcamera (Hamamatsu Photonics) controlled by SlideBook (3i) software. Wequantified EdU positive cells using the MetaMorph Image Analysissoftware (Molecular Devices). Data analyses were blinded, whereresearchers performing cell scoring were unaware of the treatmentcondition given to sample groups analyzed.

Clonal muscle stem cell proliferation and fate analyses: We assayedclonal muscle stem cell proliferation by time-lapse microscopy aspreviously described^(5,6). Briefly, we treated isolated aged MuSCs withPGE2 (Cayman Chemical) or vehicle (DMSO) for 24 hr. After five days ofgrowth on hydrogels, cells were reseeded at a density of 500 cells percm² surface area in hydrogel microwells with 600 μm diameter. Fortime-lapse microscopy we monitored cell proliferation for those wellswith single cells beginning 12 hr (day 0) to two days after seeding andrecorded images every 3 min at 10× magnification using aPALM/AxioObserver Z1 system (Carl Zeiss MicroImaging) with a customenvironmental control chamber and motorized stage. We changed mediumevery other day in between the acquisition time intervals. We analyzedtime-lapse image sequences using the Baxter Algorithms for Cell Trackingand Lineage Reconstruction to identify and track single cells andgenerate lineage trees^(5,6,8-10).

Viable and dead cells were distinguished in time-lapse sequences basedon phase-contrast boundary and motility maintenance or loss,respectively. We found that the rates of proliferation (division) anddeath in the two conditions varied over time, Therefore, we estimatedthe rates for the first and the second 24 hour intervals separately. Thevalues were estimated using the equations described in ⁶, and found inTable 1. We denote the proliferation rates in the two intervals p₂₄ andp₄₈ and the corresponding death rates d₂₄ and d₄₈. As an example, theproliferation rate in the treated condition during the second 24 hourinterval is 5.38% per hour. Table 1 (below) shows that the rates ofproliferation and death in the two conditions are similar in the firsttime interval, and that the difference in cell numbers at the end of theexperiment is due to differences in both the division rates and thedeath rates during the second time interval. The modeled cell counts inthe two time intervals are given by

${c(t)} = \{ \begin{matrix}{c_{0}{\exp ( {( {p_{24} - d_{24}} )t} )}} & {0 \leq t \leq 24} \\{{c(24)}{\exp ( {( {p_{48} - d_{48}} )( {t - 24} )} )}} & {24 < t \leq 48}\end{matrix} $

where c₀ is the number of cells at the onset. The modeled curves areplotted together with the actual cell counts in FIG. 8F.

TABLE 1 Estimated proliferation and death rates per hours. p₂₄ p₄₈ d₂₄d₄₈ DMSO 0.0488 0.0403 0.0045 0.0112 E2 0.0475 0.0538 0.0067 0.0012

The data analysis was blinded. The researchers performing the imagingacquisition and scoring were unaware of the treatment condition given tosample groups analyzed.

Quantitative RT-PCR: We isolated RNA from MuSCs using the RNeasy MicroKit (Qiagen). For muscle samples, we snap froze the tissue in liquidnitrogen, homogenized the tissues using a mortar and pestle, followed bysyringe and needle trituration, and then isolated RNA using Trizol(Invitrogen). We reverse-transcribed cDNA from total mRNA from eachsample using the SensiFAST™ cDNA Synthesis Kit (Bioline). We subjectedcDNA to RT-PCR using SYBR Green PCR Master Mix (Applied Biosystems) orTaqMan Assays (Applied Biosystems) in an ABI 7900HT Real-Time PCR System(Applied Biosystems). We cycled samples at 95° C. for 10 min and then 40cycles at 95° C. for 15 s and 60° C. for 1 min. To quantify relativetranscript levels, we used 2-ΔΔCt to compare treated and untreatedsamples and expressed the results relative to Gapdh. For SYBR GreenqRT-PCR, we used the following primer sequences: Gapdh, forward5′-TTCACCACCATGGAGAAGGC-3′, reverse 5′-CCCTTTTGGCTCCACCCT-3; Hpgd,forward 5′-TCCAGTGTGATGTGGCTGAC-3′, reverse 5′-ATTGTTCACGCCTGCATTGT-3;Ptges, forward 5′-GCTGTCATCACAGGCCAGA-3′, reverse5′-CTCCACATCTGGGTCACTCC-3; Ptges 2, forward 5′-CTCCTACAGGAAAGTGCCCA-3′,reverse 5′-ACCAGGTAGGTCTTGAGGGC-3′; Ptger1 , forward 5′GTGGTGTCGTGCATCTGCT-3′, reverse, 5′ CCGCTGCAGGGAGTTAGAGT-3′, and Ptger2,forward 5′-ACCTTCGCCATATGCTCCTT-3′, reverse 5′-GGACCGGTGGCCTAAGTATG-3′.TaqMan Assays (Applied Biosystems) were used to quantify Pax7, Myogenin,Slco2a1 (PGT), Ptger3 and Ptger4 in samples according to themanufacturer instructions with the TaqMan Universal PCR Master Mixreagent kit (Applied Biosystems). Transcript levels were expressedrelative to Gapdh levels. For SYBR Green qPCR, Gapdh qPCR was used tonormalize input cDNA samples. For Taqman qPCR, multiplex qPCR enabledtarget signals (FAM) to be normalized individually by their internalGapdh signals (VIC).

PGE2 ELISA: Muscle was harvested, rinsed in ice-cold PBS containingindomethacin (5.6 μg/ml), and snap frozen in liquid nitrogen. Frozensamples were pulverized in liquid nitrogen. The powder was transferredto an Eppendorf tube with 500 μl of lysate buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 4 mM CaCl, 1.5% Triton X-100, protease inhibitors andmicrococcal nuclease), and then homogenized using a tissue homogenizer.The PGE2 level of the supernatant was measured using a PGE2 ELISA Kit(R&D Systems, catalog #KGE004B) and expressed relative to total proteinmeasured by BCA assay (BioRad) and expressed as ng of PGE2. Each samplewas assayed in duplicate and in each of two independent experiments.

cAMP activity assay: MuSCs were treated with DMSO (vehicle) or PGE2 (10ng/ml) for 1 h and cyclic AMP levels measured according to the cAMP-GloAssay protocol optimized by the manufacturer (Promega). Each sample wasassayed in triplicate and in two independent experiments.

Flow cytometry: We assayed Annexin V as a readout of apoptosis for MuSCsafter 7 days in culture on hydrogels, after an initial acute (24 hr)treatment of vehicle (DMSO) or PGE2 (10 ng/ml). We used the FITC AnnexinV Apoptosis Detection Kit (Biolegend, cat #640914) according to theprotocol of the manufacturer. We analyzed the cells for Annexin V on aFACS LSR II cytometer using FACSDiva software (BD Biosciences) in theShared FACS Facility, purchased using an NIH S10 Shared Instrument Grant(S10RR027431-01).

Mass spectrometry—Analytes: All prostaglandin standards—PGF2α; PGE2;PGD2; 15-keto PGE2; 13,14-dihydro 15-keto PGE2; PGE2-D4; andPGF2α-D9—were purchased from Cayman Chemical. For the PGE2-D4 internalstandard, positions 3 and 4 were labeled with a total of four deuteriumatoms. For PGF2α-D9, positions 17, 18, 19 and 20 were labeled with atotal of nine deuterium atoms.

Calibration Curve preparation: Analyte stock solutions (5 mg/mL) wereprepared in DMSO. These stock solutions were serially diluted withacetonitrile/water (1:1 v/v) to obtain a series of standard workingsolutions, which were used to generate the calibration curve.Calibration curves were prepared by spiking 10 uL of each standardworking solution into 200 μL of homogenization buffer (acetone/water 1:1v/v; 0.005% BHT to prevent oxidation) followed by addition of 10 uLinternal standard solution (3000 ng/mL each PGF2α-D9 and PGE2-D4). Acalibration curve was prepared fresh with each set of samples.Calibration curve ranges: for PGE2 and 13,14-dihydro 15-keto PGE2, from0.05 ng/mL to 500 ng/mL; for PGD2 and PGF2α, from 0.1 ng/mL to 500ng/mL; and for 15-keto PGE2, from 0.025 ng/mL to 500 ng/mL.

Extraction procedure: The extraction procedure was modified from that ofPrasain et al.¹¹ and included acetone protein precipitation followed by2-step liquid-liquid extraction; the latter step enhances LC-MS/MSsensitivity. Butylated hydroxytoluene (BHT) and evaporation undernitrogen (N2) gas were used to prevent oxidation.

Solid tissues were harvested, weighed, and snap-frozen with liquidnitrogen. Muscle tissue was combined with homogenization beads and 200μL homogenization buffer in a polypropylene tube and processed in aFastPrep 24 homogenizer (MP Biomedicals) for 40 seconds at a speed of 6m/s. After homogenization, 10 μL internal standard solution (3000 ng/mL)was added to tissue homogenate followed by sonication and shaking for 10minutes. Samples were centrifuged and the supernatant was transferred toa clean eppendorf tube. 200 μL hexane was added to the sample, followedby shaking for 15 minutes, then centrifugation. Samples were frozen at−80° C. for 40 minutes. The hexane layer was poured off from the frozenlower aqueous layer, and discarded. After thawing, 25 μL of 1N formicacid was added to the bottom aqueous layer, and the samples werevortexed. For the second extraction, 200 μL chloroform was added to theaqueous phase. Samples were shaken for 15 minutes to ensure fullextraction. Centrifugation was performed to separate the layers. Thelower chloroform layer was transferred to a new eppendorf tube andevaporated to dryness under nitrogen at 40° C. The dry residue wasreconstituted in 100 μL acetonitrile/10 mM ammonium acetate (2:8 v/v)and analyzed by LC-MS/MS.

LC-MS/MS: Since many prostaglandins are positional isomers withidentical masses and have similar fragmentation patterns,chromatographic separation is critical. Two SRM transitions—onequantifier and one qualifier—were carefully selected for each analyte.Distinctive qualifier ion intensity ratios and retention times wereessential to authenticate the target analytes. All analyses were carriedout by negative electrospray LC-MS/MS using an LC-20AD_(XR) prominenceliquid chromatograph and 8030 triple quadrupole mass spectrometer(Shimadzu). HPLC conditions: Acquity UPLC BEH C18 2.1×100 mm, 1.7 umparticle size column was operated at 50° C. with a flow rate of 0.25mL/min. Mobile phases consisted of A: 0.1% acetic acid in water and B:0.1% acetic acid in acetonitrile. Elution profile: initial hold at 35% Bfor 5 minutes, followed by a gradient of 35%-40% in 3 minutes, then40%-95% in 3 minutes; total run time was 14 minutes. Injection volumewas 20 uL. Using these HPLC conditions, we achieved baseline separationof the analytes of interest.

Selected reaction monitoring (SRM) was used for quantification. The masstransitions were as follows: PGD2: m/z 351.10→m/z 315.15 (quantifier)and m/z 351.10→m/z 233.05 (qualifier); PGE2: m/z 351.10→m/z 271.25(quantifier) and m/z 351.10→m/z 315.20 (qualifier); PGF2α: m/z353.10→m/z 309.20 (quantifier) and m/z 353.10→m/z 193.20 (qualifier); 15keto-PGE2: m/z 349.30→m/z 331.20 (quantifier) and m/z 349.30→m/z 113.00(qualifier); 13, 14-dihydro 15-keto PGE2: m/z 351.20→m/z 333.30(quantifier) and m/z 351.20→m/z 113.05 (qualifier); PGE2-D4: m/z355.40→m/z 275.20; and PGF2α-D9: m/z 362.20→m/z 318.30. Dwell time was20-30 ms.

Quantitative analysis was done using LabSolutions LCMS (Shimadzu). Aninternal standard method was used for quantification: PGE2-D4 was usedas an internal standard for quantification of PGE2, 15-keto PGE2, and13, 14-dihydro 15-keto PGE2. PGF2α-D9 was the internal standard forquantification of PGD2 and PGF2α. Calibration curves were linear(R>0.99) over the concentration range using a weighting factor of 1/X²where X is the concentration. The back-calculated standardconcentrations were ±15% from nominal values, and ±20% at the lowerlimit of quantitation (LLOQ).

In vivo muscle force measurement: Aged mice (18 mo.) were subjected todownhill treadmill run for 2 consecutive weeks. During week 1, mice randaily for 5 days and rested on days 6 and 7. Two hours after eachtreadmill run during week 1, each (lateral and medial) gastrocnemius(GA) muscle from both legs of each mouse was injected with a dose ofeither PBS (vehicle control) or 13 nM dmPGE2 (experimental group).During week 2, mice were subjected to 5 days treadmill run only. Thetreadmill run was performed using the Exer3/6 (Columbus Instruments).Mice ran for 10 minutes on the treadmill at 20 degrees downhill,starting at a speed of 7 meters/min. After 3 min, the speed wasincreased by 1 meter/min to a final speed of 14 meter/min. 10 minutesrun time was chosen, as exhaustion defined as the inability of theanimal to remain on the treadmill despite electrical prodding, wasobserved at a median of 12 minute in an independent control aged mousegroup. Force measurements were on the GA muscles at week 5 based on aprotocol published previously⁵. Briefly, for each mouse, an incision wasmade to expose the GA. We severed the calcaneus bone with intactachilles tendon and attached the tendon-bone complex to a 300C-LR forcetransducer (Aurora Scientific) with a thin metal hook. The muscles andtendons were kept moist by periodic wetting with saline (0.9% sodiumchloride) solution. The lower limb was immobilized below the knee by ametal clamp without compromising the blood supply to the leg. The mousewas under inhaled anesthetic (2% isofluorane) during the entire forcemeasuring procedure and body temperature was maintained by a heat lamp.In all measurements, we used 0.1-ms pulses at a predeterminedsupramaximal stimulation voltage. The GA muscles were stimulated via theproximal sciatic nerve using a bipolar electrical stimulation cuffdelivering a constant current of 2 mA (square pulse width 0.1 ms). GAmuscles were stimulated with a single 0.1-ms pulse for twitch forcemeasurements, and a train of 150 Hz for 0.3 s pulses for tetanic forcemeasurements. We performed five twitch and then five tetanicmeasurements on each muscle, with 2-3 min recovery between eachmeasurement with n=5 mice per group. Data were collected with a PCI-6251acquisition card (National Instruments) and analyzed in Matlab. Wecalculated specific force values by normalizing the force measurementsby the muscle physiological cross-sectional areas (PCSAs), which weresimilar between the control and the experimental PGE2 treated group(Table 2). PCSA (measured in mm²) was calculated according to thefollowing equation¹²:

PCSA (mm²)=[mass (g)×Cos θ]÷[ρ(g/mm³)×fiber length (mm)],

where θ is pennation angle of the fiber and ρ is muscle density(0.001056 g/mm³).

Statistical analyses: We performed cell culture experiments in at leastthree independent experiments where three biological replicates werepooled in each. In general, we performed MuSC transplant experiments inat least two independent experiments, with at least 3-5 totaltransplants per condition. We used a paired t-test for experiments wherecontrol samples were from the same experiment in vitro or fromcontralateral limb muscles in vivo. A non-parametric Mann-Whitney testwas used to determine the significance difference between untreated (−)vs treated (PGE or dmPGE2) groups using a=0.05. ANOVA or multiple t-testwas performed for multiple comparisons with significance leveldetermined using Bonferroni correction or with Fisher's test asindicated in the figure legends. Unless otherwise described, data areshown as the mean±s.e.m.

Methods references: Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S. &Blau, H. M. Self-renewal and expansion of single transplanted musclestem cells. Nature 456, 502-506, doi:10.1038/nature07384 (2008);Schneider, A. et al. Generation of a conditional allele of the mouseprostaglandin EP4 receptor. Genesis 40, 7-14, doi:10.1002/gene.20048(2004); Murphy, M. M., Lawson, J. A., Mathew, S. J., Hutcheson, D. A. &Kardon, G. Satellite cells, connective tissue fibroblasts and theirinteractions are crucial for muscle regeneration. Development 138,3625-3637, doi:10.1242/dev.064162 (2011); Safran, M. et al. Mousereporter strain for noninvasive bioluminescent imaging of cells thathave undergone Cre-mediated recombination. Molecular imaging 2, 297-302(2003); Cosgrove, B. D. et al. Rejuvenation of the muscle stem cellpopulation restores strength to injured aged muscles. Nature medicine20, 255-264, doi:10.1038/nm.3464 (2014); Gilbert, P. M. et al. Substrateelasticity regulates skeletal muscle stem cell self-renewal in culture.Science 329, 1078-1081, doi:10.1126/science.1191035 (2010); Papapetrou,E. P. et al. Genomic safe harbors permit high beta-globin transgeneexpression in thalassemia induced pluripotent stem cells. Naturebiotechnology 29, 73-78, doi:10.1038/nbt.1717 (2011); Chenouard, N. etal. Objective comparison of particle tracking methods. Nature methods11, 281-289, doi:10.1038/nmeth.2808 (2014); Magnusson, K. E., Jalden,J., Gilbert, P. M. & Blau, H. M. Global linking of cell tracks using theViterbi algorithm. IEEE transactions on medical imaging 34, 911-929,doi:10.1109/TMI.2014.2370951 (2015); Maska, M. et al. A benchmark forcomparison of cell tracking algorithms. Bioinformatics 30, 1609-1617,doi:10.1093/bioinformatics/btu080 (2014); Prasain, J. K., Hoang, H. D.,Edmonds, J. W. & Miller, M. A. Prostaglandin extraction and analysis inCaenorhabditis elegans. Journal of visualized experiments: JoVE,doi:10.3791/50447 (2013); Burkholder, T. J., Fingado, B., Baron, S. &Lieber, R. L. Relationship between muscle fiber types and sizes andmuscle architectural properties in the mouse hindlimb. J Morphol 221,177-190, doi:10.1002/jmor.1052210207 (1994).

TABLE 2 Physiological cross-sectional area (PCSA) of aged gastrocnemiusweek 5 post-exercise. PCSA Pennation Fiber GA (medial + angle θ lengthMass lateral) Mouse ID Leg (degree) Cosine(θ) (mm) (g) (mm²) Control-1Left 21 0.93 6.88 0.18 23.13 Right 21 0.93 6.64 0.18 23.82 Control-2Left 26 0.90 4.03 0.16 33.79 Right 22 0.93 5.34 0.16 26.31 Control-3Left 21 0.93 4.52 0.15 29.34 Right 23 0.92 4.59 0.17 32.28 Control-4Left 24 0.91 5.07 0.14 23.89 Right 23 0.92 4.75 0.13 23.86 Control-5Left 19 0.95 6.07 0.16 17.75 Right 18 0.95 6.05 0.15 10.25 dmPGE2-1 Left12 0.98 7.60 0.25 30.47 Right Tendon — — — — damage dmPGE2-2 Left 120.96 4.85 0.16 30.56 Right 16 0.91 4.80 0.14 26.55 dmPGE2-3 Left 14 0.975.89 0.17 26.52 Right 13 0.94 5.63 0.14 22.94 dmPGE2-4 Left 14 0.97 6.670.14 19.29 Right 13 0.97 7.74 0.16 19.07 dmPGE2-5 Left 11 0.98 5.56 0.1728.42 Right 11 0.98 5.54 0.16 26.85 Avg. Control 25.09 Avg. dmPGE2 25.63

Example 2 Increased Muscle Forces After Prostaglandin E2 (PGE2)Injection

This example shows an increase in specific twitch force of gastrocnemiusmuscles in aged mice injected with PGE2. The aged mice (18 months old)were subject to treadmill run to exhaustion daily for 10 days. Thetreadmill run was performed using the Exer3/6 (Columbus Instruments).Mice ran on the treadmill at 20 degrees downhill, starting at a speed of10 meters/min. After 3 min, the speed was increased 1 meter/min to afinal speed of 20 meters/min. Exhaustion was defined as the inability ofthe animal to remain on the treadmill despite electrical prodding. 2 hafter each treadmill run, both gastrocnemius muscles of each mouse wereinjected with either PBS (control group) or 3 nM PGE2 (experimentalgroup). The force measurement was performed 4 weeks after the lasttreadmill run using a 300C-LR force transducer (Aurora Scientific) witha single 0.1 ms pulse at predetermined supramaximal stimulationintensity.

Representative raw muscle force traces of single gastrocnemius musclesare provided in FIGS. 5M-5N. The muscle force and synchronization pulseswere recorded via a PCI-6251 acquisition card (National Instruments) andanalyzed using Matlab. FIGS. 5O-5P show the specific muscle force valuesthat were calculated by normalizing the force measurements with themuscle physiological cross-sectional area. The specific twitch forcevalues (kN/m²) are represented by the Box and Whiskers plot that showsthe minimum, maximum, and median values. Five repetitive measurementswere made from each muscle. N=4 for the control group and n=5 for thePGE2 injected group. ** represents a statistical significant value ofp<0.005 by 2-tailed Mann Whitney test.

FIGS. 5Q and 5R show twitch force and tetanic force data, respectively,from a separate experiment in which mice were treated with PGE2 orvehicle only. Importantly, we observed an increase in isometric force inaged (18-22 mo) mice injected with PGE2 and subjected to downhilltreadmill exercise. Briefly, aged mice ran daily (at 20 degrees downhilland 14 meter/min maximum speed for 10min) for 5 days and rested on days6 and 7. This eccentric exercise regime leads to MuSC expansion due to acycle of muscle degeneration and regeneration. Two hours after eachtreadmill run during week 1, TA muscles of both legs of each mouse wereinjected with either PBS (vehicle control group) or 10 μg PGE2(experimental group). During week 2, mice were subjected to 5 daystreadmill run only. Force measurements (twitch and tetanic) wereperformed on the TA muscles at week 5 using our previously publishedprotocols. The PGE2 treated group exhibited a significant increase inforce compared to the control group.

Example 3 Prostaglandin E2 is Essential for Efficacious Skeletal MuscleStem Cell Function, Augmenting Regeneration and Strength

Skeletal muscles harbor quiescent muscle-specific stem cells (MuSCs)capable of tissue regeneration throughout life. Muscle injuryprecipitates a complex inflammatory response in which a multiplicity ofcell types, cytokines and growth factors participate, includingprostaglandins. Here we show that Prostaglandin E2 (PGE2) directlytargets MuSCs via the EP4 receptor leading to MuSC expansion. An acutetreatment with PGE2 suffices to robustly augment muscle regeneration byeither endogenous or transplanted MuSCs. Loss of PGE2 signaling byspecific genetic ablation of the EP4 receptor in MuSCs impairsregeneration leading to decreased muscle force. Inhibition of PGE2production through NSAID administration just after injury similarlyhinders regeneration and compromises muscle strength. Mechanistically,the PGE2 EP4 interaction causes MuSC expansion by triggering a cyclicAMP/phosphoCREB pathway that activates the proliferation-inducingtranscription factor, Nurr1. Our findings reveal that loss of PGE2signaling to MuSCs during recovery from injury impedes muscle repair andstrength. Through such gain or loss of function experiments, we foundthat PGE2 signaling acts as a rheostat for muscle stem cell function.Decreased PGE2 signaling due to NSAIDs or increased PGE2 due toexogenous delivery dictates MuSC function which determines the outcomeof regeneration. The markedly enhanced and accelerated repair of damagedmuscles following intramuscular delivery of PGE2 suggests a newindication for this therapeutic agent.

Muscle repair after injury entails an immune response that orchestratesefficacious regeneration. Here we identify Prostaglandin E2 (PGE2) as acrucial inflammatory mediator of muscle stem cells (MuSCs), the buildingblocks of muscle regeneration. PGE2 is synthesized and secreted into thestem cell niche in response to injury leading to robust MuSCproliferation, key to myofiber repair. EP4 is the receptor that mediatesPGE2 signaling in MuSCs and genetically engineered mice that lack EP4 inMuSCs have impaired regeneration. Non-steroidal anti-inflammatory drugs(NSAIDs), commonly used to treat pain after muscle injury, inhibit PGE2synthesis, hinder muscle regeneration, and lead to weakened muscles.Importantly, a single treatment of injured muscles with PGE2dramatically accelerates muscle repair and recovery of strength.

Satellite cells, also known as muscle stem cells (MuSCs) are crucial tomuscle regeneration. They reside in a quiescent state in nichesjuxtaposed to myofibers in muscle tissues, poised to respond to damageand repair skeletal muscles throughout life (1-4). Muscle injuryprecipitates an inflammatory response that is marked by the sequentialinfiltration of multiple cell types including neutrophils, monocytes,macrophages, T-cells and fibroadipocytes, and is accompanied by musclestem cell activation. During this inflammatory phase, concurrent wavesof cytokine and growth factor release, including CC-chemokine ligand 2(CCL2), IL-10, IL-1β, tumor necrosis factor-α (TNFα), transforminggrowth factor-β1 (TGFβ1) (3, 5-10). In addition, prostaglandins, potentlipid mediators of inflammation, are synthesized and secreted by immuneand myogenic cells (6, 11). Prostaglandins derive from arachidonic acid,which is released from membrane phospholipids by phospholipase A2 andconverted by cyclooxygenase enzymes (COX-1 and -2) into prostaglandin H2(PGH2), and subsequently into the different prostaglandin subtypes,PGD2, PGE2, PGF2α, PGI2 or thromboxane (TXA2). Specific to thegeneration of PGE2 are the prostaglandin synthases (PGES: mPGES-1,mPGES-2 and cPGES) (11-13).

While PGE2 has been associated with muscle regeneration, it was notknown to have a direct beneficial effect on muscle regeneration andstrength until this benefit was discovered by the inventors of thepresent invention. Conflicting reports suggest that PGE2 can eitherpromote myoblast proliferation or differentiation in culture (14-18). Inthe COX2-knockout mouse model, which lacks PGE2, regeneration isdelayed. However, the mechanism by which PGE2 acts could not beestablished in these studies due to the systemic constitutive loss ofCOX2 and consequent nonspecific effects on many cell types (15, 19).Similarly, muscle recovery after injury was impaired in mice given aCOX-2 inhibitor (15). Additionally, mice treated with non-steroidalanti-inflammatory drugs (NSAIDs), which block the production ofprostaglandins through inhibition of COX1 and COX2, exhibitedregeneration deficits (20, 21). Moreover, NSAIDS lead to an attenuationof exercise-induced expansion of human satellite cells in biopsies (20).Likewise, glucocorticoids, which reduce prostaglandin synthesis bysuppressing phospholipase A2, COX-2 and mPGES-1 expression, adverselyaffect the recovery of muscle strength in polymyositis patients (22).However, since the target of NSAIDs and glucocorticoids are the COXenzymes, this effect could entail a number of prostaglandin subtypes inaddition to PGE2 and therefore have pleiotropic effects. Thus, to datethe spatiotemporal effects of PGE2 in muscle regeneration remainunresolved. Moreover, although inhibition of PGE2 synthesis and activitywas shown to be detrimental to the recovery of muscle function, thestudies referenced here do not provide any suggestion thatadministration of PGE2 could be directly beneficial for muscleregeneration and the recovery of muscle function.

The inventors have discovered that in response to injury, PGE2 istransiently induced in muscle tissues. To establish if PGE2 actsdirectly on MuSCs, the building blocks of muscle regeneration, wegenerated mice in which the PGE2 receptor, EP4, could be conditionallyablated in MuSCs. In addition, we established transgenic reporter micethat enabled specific tracking of MuSC contribution to regenerationdynamically and sensitively over time by bioluminescence imaging afterPGE2 delivery. We coupled these models with assays of muscle force andfound a direct link between the ability of MuSCs to respond to PGE2 andregeneration, leading to restoration of force. Gain and loss of functionexperiments revealed that PGE2 signaling acts as a rheostat for musclestem cell function. We provide evidence that although PGE2 is normallysynthesized after injury, by transiently increasing PGE2 levels abovenormal endogenous levels, regeneration is augmented. Our data indicatethat PGE2 impacts regeneration and has therapeutic applications.

A surge of PGE2 in damaged muscle tissues accelerates MuSCproliferation: We sought to identify an activator of MuSC function bycapitalizing on an inflammatory response that mediates muscleregeneration. Since muscle injury triggers an immediate inflammatoryresponse (5, 7, 8, 23), we hypothesized that a transiently inducedinflammatory modulator could regulate MuSC function and play a crucialrole in regeneration. We performed qRT-PCR and detected increased levelsof the Ptger4 receptor (EP4) for PGE2, a potent lipid mediator duringacute inflammation (11), on isolated MuSCs obtained by dissociatingmuscle tissue followed by fluorescence activated cell sorting (FACS)(FIG. 12A). In accordance with receptor expression, we detected a surgein the levels of PGE2 in mouse muscle lysates three days after injury bystandard paradigms entailing notexin injection or cryoinjury (FIGS. 12B,12C, and 18A). The concomitant transient upregulation of itssynthesizing enzymes, Ptges and Ptges2 was also detected (FIG. 12D).Although other cell types within muscles may also produce PGE2 inresponse to injury such as endothelial cells, inflammatory cells andFAPs, the myofibers that circumscribe MuSCs are a source of PGE2, asobserved in conditioned medium from dissociated myofibers (FIG. 12E).Moreover, upon treatment of myofibers with indomethacin, a NSAID thatinhibits COX2, PGE2 synthesis is markedly reduced (FIG. 12E). The peakin PGE2 levels coincides temporally with the expansion of MuSCs and thewell documented accumulation of inflammatory cytokines such as TGFβ1,CCL2, IL-10, IL-1β and TNFα post-injury, where MuSC activation andexpansion takes place (3, 5, 7, 8). Although PGE2 has previously beenimplicated in the inflammatory damage response, the cellular andmolecular mechanism by which it acts in muscle regeneration has yet tobe resolved.

To determine whether PGE2 has a direct effect on MuSC expansion, weassessed the proliferation potential of FACS isolated MuSCs (24) treatedwith PGE2 (10 ng/ml) in culture. This concentration of PGE2 was selectedbased on a dose-response assay, which resolved the lowest drugconcentration that promotes a robust MuSC proliferation response (FIG.18B). We found that a 1-day exposure to PGE2 in culture induced a 6-foldincrease in the number of MuSCs relative to controls one week later(FIG. 12F). This increase in cell division after PGE2 treatment was alsoevident by EdU incorporation (FIGS. 18C and 18D). Culture of MuSCs inmedia with charcoal stripped serum, which is depleted of lipidcomponents including prostaglandins (25), markedly impeded cellproliferation. Addition of PGE2 rescued this block in proliferation(FIG. 18E). Notably, whereas freshly isolated MuSCs expressed relativelyhigh levels of EP4 receptor mRNA, expression progressively declined tonegligible levels was as the cells gave rise to increasinglydifferentiated muscle cells in culture. This result suggests that MuSCsare the myogenic cell type most responsive to PGE2 (FIG. 12A). Wefurther analyzed the effect of PGE2 at the single cell level by trackingindividual MuSCs by time-lapse microscopy analysis in a hydrogel‘microwell’ platform as previously described (26, 27) (FIGS. 12G-K and18F-H). Clonal assays can reveal differences that are obscured byanalysis of the population as a whole. Data were collected over a 38 htime period and then analyzed using the Baxter Algorithms for CellTracking and Lineage Reconstruction (26-28). We observed a markedincrease in cumulative cell divisions and cell numbers in response toPGE2, spanning 6 generations for the most robust clones (FIGS. 12G and12H). The basis for the difference between PGE2-treated cells andvehicle-treated controls is that immediately following PGE2 additionpost-plating, entry into mitosis is accelerated which is the cause ofthe subsequent increased expansion (FIGS. 121, 12J, 18G, and 18H). Thesubsequent exponential increase in cells in both conditions exacerbatesthe difference at the onset, culminating in almost twice the number oftotal cells at the end of the 38 h timespan (FIGS. 18G and 18H). Theconcomitant increase in the incidence of larger cell sizes observedafter PGE2 treatment (FIG. 12K), support its role in mitotic events(29).

PGE2 treatment augments muscle regeneration: To determine if PGE2impacted the function of MuSCs in regeneration, we performed in vivoexperiments. To monitor the dynamics of regeneration over time in aquantitative manner, we capitalized on a sensitive and quantitativebioluminescence imaging (BLI) assay we previously developed formonitoring MuSC function post-transplantation (24, 26, 27, 30). MuSCswere isolated from transgenic mice expressing both GFP and luciferase(GFP/Luc mice) and equivalent numbers of MuSCs (250 cells) werecoinjected with either PGE2 or vehicle only into injured TAs of NOD-SCIDgamma (NSG) mice. PGE2 coinjection enhanced the regenerative capacity ofMuSCs by nearly two orders of magnitude compared to controls assessed byBLI. Histological analysis reveals GFP+MuSC engraftment in the niche andGFP⁺ fibers resulting from fusion over the time course (FIGS. 13A, 19A,and 19B). Moreover, following engraftment, a secondary injury elicited aspike in BLI signals of PGE2-treated MuSCs relative to controls,suggesting enhanced stem cell repopulation (FIG. 13A). Notably PGE2 isknown to have a relatively short half-life in vivo (31). Thus theseexperiments demonstrate that transient exposure of MuSCs to PGE2 at thetime of co-delivery to injured muscle suffices to significantly enhancemuscle regeneration.

We postulated that delivery of PGE2 alone could increase endogenous MuSCnumbers and enhance regeneration, circumventing the need for a celltherapeutic. We reasoned that PGE2 delivered during the early timewindow immediately post-injury could augment the beneficial effects ofthe innate inflammatory response and PGE2 surge. To test thispossibility, muscles of young mice were injured and three days later weinjected a bolus of PGE2 (FIG. 13B). We observed a striking increase(65±7%) in endogenous PAX7-expressing MuSCs in the classic satellitecell niche beneath the basal lamina and atop myofibers 14 dayspost-injury (FIGS. 13B and 13C). PGE2 is only effective after injury, asno difference from vehicle-injected controls was observed in the absenceof tissue damage (data not shown). A striking shift in the distributionof myofibers from smaller toward larger sizes, assessed ascross-sectional-area was evident over the time course of regeneration(FIGS. 13D, 13E, 19A, and 19B). This change reflects the remodeling ofmyofiber architecture that accompanies the observed acceleratedregeneration, as muscle mass did not increase during this time period(FIG. 19C). In addition, we tracked the response to injury and PGE2 ofendogenous MuSCs by luciferase expression using a transgenic mousemodel, Pax7^(CreERT2);Rosa26-LSL-Luc (FIGS. 13F and 13G). The BLI datawere in agreement with the histological data (FIGS. 13B and 13C). That asingle injection of PGE2 post-injury could suffice to boost endogenousMuSC numbers and regenerative function leading to this degree ofaccelerated regeneration was quite unexpected.

EP4 receptor mediates PGE2 signaling to promote MuSC proliferation andengraftment: PGE2 is known to signal through four G-protein coupledreceptors (Ptger1-4; EP1-4) (6, 11), but the expression of thesereceptors in MuSCs has not previously been reported. An analysis of thetranscript levels of the different receptors (Ptger1-4) revealed that 24h after PGE2 treatment, the most highly expressed receptor in MuSCs isPtger4 (FIG. 14A). PGE2 treated MuSCs showed elevated downstreamintracellular cyclic AMP (cAMP) levels (FIG. 14B), a response associatedto EP4 signaling (11), and in the presence of an EP4 antagonist,ONO-AE3-208, the increased proliferation response induced by PGE2 wasblunted (FIG. 14C). This data confirms that PGE2 signals through the EP4receptor to promote proliferation. The specificity of PGE2 for EP4 wasmost clearly shown by the marked reduction in proliferation of MuSCslacking the receptor following Cre-mediated conditional ablation of EP4in MuSCs isolated from EP4^(f/f) mice (FIGS. 14D and 20A-D). Arequirement for EP4 in the proliferative response to PGE2 was confirmedby tamoxifen treatment of MuSCs isolated from Pax7^(CreERT2):EP4^(f/f)mice in which Cre-mediated EP4 ablation is under the control of theMuSC-specific Pax7 promoter (FIGS. 20E and 20F). Notably, compensationby other PGE2 receptors does not occur in MuSCs lacking EP4 asexpression of EP1, EP2 and EP3 receptors (Ptger 1-3) remains low inMuSCs (FIG. 20G). Together, these data show that PGE2 and its receptorEP4 are crucial for MuSC proliferation. To determine if EP4 plays a rolein MuSC function in vivo, we transplanted luciferase-expressing MuSCsthat lacked the EP4 receptor following conditional ablation in cultureinto injured TAs of NSG mice. The BLI signal that was initially detectedprogressively declined to levels below the threshold of significance(FIGS. 14E and 20A-D). Thus, in the absence of PGE2 signaling via theEP4 receptor regeneration is impaired.

Transcription factor Nurr1 is a downstream mediator of PGE2/EP4signaling in MuSCs: To perform an unbiased search for mediators ofsignaling downstream of PGE2 that mediate the enhanced effect of MuSCfunctions, we performed an RNA-seq analysis comparing isolated MuSCstreated with vehicle (control) or PGE2 for 24 h (FIG. 21A).Bioinformatics analyses using Ingenuity Pathway Analysis (IPA) andMetacore software packages revealed that in addition to regulators ofPGE2 metabolism, PGE2 treatment of MuSCs led to an increase in molecularand cellular functions consistent with stem cell expansion, includingcAMP signaling, and cell cycle regulation (FIGS. 21B and 21C). Among thetop 200 differentially expressed genes with a non-adjusted p-value<0.05,only 11 transcription factors were identified (FIG. 15A). Nurr1 wasamong the few that were differentially expressed. Nurr1 had alsopreviously been shown to mediate PGE2 signaling through cAMP andphospho-CREB to induce cell proliferation in colorectal cancer andneuronal cells (32, 33). To investigate its putative role as adownstream effector of EP4 signaling in MuSCs, we examined itsexpression in vivo. Remarkably, the time window of Nurr1 expressionmirrored that of PGE2 in muscle tissue, peaking at day 3 post-injury(FIGS. 15B and 12B). In culture, PGE2 treatment increased Nurr1 mRNA andprotein expression (FIGS. 15C and 15D) and Nurr1 knockdown blunted theeffect of PGE2 in inducing MuSC proliferation (FIGS. 4E and 21D). Todetermine the specificity of Nurr1 transcriptional regulation to PGE2mediated-EP4 receptor signaling we ablated the EP4 receptor inPax7^(CreERT2):EP4^(f/f) MuSCs by tamoxifen treatment (FIG. 21E). Nurr1was not unregulated after PGE2 treatment in EP4 knockout MuSCs (FIG.15F). Expression of Nurr1 was highest in MuSCs and declined at the onsetof differentiation of myogenic cells, in accordance with the expressionpattern of EP4 (FIG. 15G). Together, these data implicate the Nurr1transcription factor as a mediator of PGE2/EP4 signaling that triggersMuSC expansion.

Loss of PGE2 signaling impairs muscle regeneration and strength: Todetermine if EP4 is required for regeneration in vivo, we used thePax7^(CreERT2):EP4^(f/f) mouse model in which EP4 is specifically andconditionally ablated in MuSCs by sequential intraperitoneal tamoxifeninjection into mice (FIG. 16A). Induction of EP4 ablation was highlyefficient in Pax7⁺ cells in vivo following tamoxifen treatment andinjury. Ptger4 mRNA levels detected in sorted MuSCs was 96% lower thanin the control (FIGS. 16A and 16B). In the absence of EP4 signaling inMuSCs, we observed an aberrant persistence of immature centrallynucleated regenerating myofibers that express embryonic myosin heavychain (eMyHC) at day 7 post-injury (FIGS. 16C and 16E). This evidence ofimpaired regeneration was corroborated by a shift toward myofibers withdiminished myofiber cross sectional area relative to controls at day 21post-injury (FIGS. 16D and 16E). In these experiments, PGE2 can act onother cell types in muscle tissue in the course of regeneration, such asmature myofibers, fibroadipogenic progenitors (FAPs) and immune cells;however, these cells were not sufficient to restore the EP4-deficientMuSC functions. These features provide strong evidence that in theabsence of EP4 signaling efficacious muscle regeneration is impeded.

We further tested whether the defects in muscle repair stemming fromspecific loss of EP4 in MuSCs impacted muscle strength. Strikingly,eliminating signaling through EP4 on MuSCs alone led to a 35±6% and31±4% decrease in twitch and tetanus force, respectively (FIGS. 16F-H),without apparent loss of muscle mass (FIG. 22A). To determine if theabsence of PGE2 altered muscle regeneration and strength after injury,we subjected mice to treatment with a non-steroidal anti-inflammatorydrug (NSAID, indomethacin). A single indomethacin injection into TAmuscles of a Pax7^(CreERT2);Rosa 26-L SL-Luc mouse model three daysafter injury led to a decline in BLI relative to controls, indicative ofan impairment in muscle stem cell expansion and regeneration (FIGS. 16Iand 16J). This loss of regenerative capacity after NSAID treatment wasaccompanied by a substantial 33±7% reduction in twitch force compared tocontrols (FIGS. 16K, 16L, and 22B). The diminished strength seen uponglobal muscle inhibition of PGE2 synthesis mirrored that observed inregenerating muscle with MuSC-EP4 specific knockout, suggesting thatMuSC expansion accounts for the majority of the PGE2 mediated effects onmuscle regeneration.

We have discovered that a major effect of PGE2 during muscleregeneration is to target MuSCs directly. PGE2 has been implicated as animmunomodulator that acts on neutrophils, mast cells, and macrophagesthat are crucial to the early inflammatory response after injury. Theensuing cytokine storm is thought to induce muscle stem cell function inregeneration (3, 6, 7, 11). Studies in whole body COX2 KO mice, in whichall prostaglandin synthesis was ablated, supported this conclusion (15,19). Myoblasts have been proposed as the cell type responsive to PGE2 inculture (14, 16-18, 34, 35), but these cells perform poorly inregeneration (24) and cannot account for the observed effects. Moreover,other studies implicating PGE2 in regeneration all suffered frompleiotropic effects on a multiplicity of cell types.

MuSCs are crucial to development and regeneration (1-3, 24, 36-38) andtheir numbers dramatically increase in response to insults that damagethe muscles in mice and humans (5, 20, 39-42). Injections of MuSCs intoinjured muscles leads to their exponential increase, whereas injectionof their myoblast derivatives results in a decline in numbers, revealinga remarkable distinction in regenerative capacity of these two celltypes (24). Here we show that the major effect of PGE2 during muscleregeneration is on MuSCs and that this effect is direct and mediated bythe EP4 receptor. Notably, EP4 is robustly expressed on MuSCs andprogressively diminishes to negligible levels on differentiatingmyoblasts suggesting that the most responsive myogenic cell type to PGE2is the MuSC. Mechanistically, once PGE2 engages the EP4 receptor, itactivates cAMP and the downstream proliferation-inducing transcriptionfactor Nurr1 leading to accelerated MuSC proliferation (FIG. 17).Although Nurr1 has been associated in intestinal epithelial cells withinduction of proliferation and regeneration by directly blocking thecell cycle inhibitor p21 (Waf1/Cip1) in intestinal epithelial cells(43), its role in the expansion of stem cells, and particularly musclestem cells, has not previously been described. The finding that furthersubstantiates that Nurr1 mediates the onset of MuSC proliferation invivo is that its levels transiently peak in muscle tissues three dayspost injury, concomitant with the observed surge in PGE2.

We show that MuSC function and engraftment are strictly dependent onPGE2 signaling through its receptor by its conditional and specificablation of EP4 using two approaches. Ablation of EP4 on MuSCs in vitrofollowed by transplantation in vivo leads to diminished engraftmentevident by BLI. The most striking evidence of a crucial role for EP4derives from its in vivo ablation of EP4 specifically on endogenousMuSCs which causes a marked reduction in muscle strength post-injuryaccompanied by a shift toward smaller and more immature myofibersrelative to controls (FIG. 17). Thus, in the absence of the EP4receptor, regeneration by both transplanted and endogenous MuSCs isseverely impaired.

The surge in PGE2 post-injury is transient. Similarly, acute PGE2treatment enhances and accelerates muscle regeneration long-term. Whenfreshly isolated MuSCs were coinjected with PGE2 into injured muscles, aboost in muscle repair was evident by BLI. A single ex vivo exposure ofhematopoietic stem cells to PGE2 had a similarly pronounced effect onsubsequent stem cell expansion and reconstitution of the bloodpost-transplant (44). Indeed, a single injection of PGE2 alone (withoutMuSCs) directly into injured muscles led to a striking increase inendogenous MuSC numbers and myofiber sizes that was apparent within 2weeks. The beneficial effects of delivery of the inhibitor of thePGE2-degrading enzyme (15-PGDH), SW033291, on hematopoietic, liver, andcolon regeneration are likely due to a similar augmentation ofendogenous PGE2 levels (45). Notably, PGE2 and its analogues have safelybeen used in patients for more than a decade, for instance to inducelabor (46) and to promote hematopoietic stem cell transplantation (44).Together with our findings, these studies pave the way for its clinicaluse in boosting muscle repair post-injury.

We show that PGE2 levels act as a rheostat that controls the efficacy ofregeneration. Augmentation of the innate pro-inflammatory PGE2 responseto injury leads to accelerated MuSC expansion and muscle regeneration.By contrast, NSAID administration at the time of injury to control pain,a common practice, abrogates that effect, suggesting that PGE2 signalingduring this early temporal window is crucial to its beneficial effects.Most striking is our finding that a single PGE2 treatment suffices torapidly and robustly invoke a muscle stem cell response to enhanceregeneration of damaged muscle and restore strength.

We performed all experiments and protocols in compliance with theinstitutional guidelines of Stanford University and Administrative Panelon Laboratory Animal Care (APLAC). We obtained young wild-type C57BL/6mice from Jackson Laboratory. Double-transgenic GFP/luc mice wereobtained as described previously (Jackson Laboratory, Stock #008450)(24). NOD-scid gamma (NSG) were obtained from Jackson Laboratory (Stock#0055570). EP4^(flox/flox) (EP4^(f/f)) mice were a kind gift from K.Andreasson (Stanford University) (Jackson Laboratories, Stock #028102)(47). Double-transgenic Pax7^(CreERT)2;EP4^(f/f) were generated bycrossing Pax7^(CreERT2) mice obtained from Jackson Laboratory (Stock#017763) (48) and EP4^(f/f) mice. Double-transgenicPax7^(CreERT2);Rosa26-LSL-Luc were generated by crossing Pax7^(CreERT2)mice and Rosa26-LSL-Luc obtained from Jackson Laboratory (Stock #005125)(49). We validated these genotypes by appropriate PCR-based strategies.All mice from transgenic and wild-type strains were of young age (2-4months). All experiments were conducted using age and gender-matchedcontrols, and littermates randomly assigned to experimental groups.

We used an injury model entailing intramuscular injection of 10 μl ofnotexin (10 μg ml⁻¹; Latoxan, catalog #L8104) or cardiotoxin (10 μM;Latoxan, catalog #L8102) into the Tibilais anterior (TA) muscle. Forcryoinjury, an incision was made in the skin overlying the TA muscle anda copper probe, chilled in liquid nitrogen, was applied to the TA musclefor three 10 s intervals, allowing the muscle to thaw between eachapplication of the cryoprobe. When indicated, 48 hr after injury either16,16-Dimethyl Prostaglandin E2 (dmPGE2) (13 nmol, Tocris, catalog#4027), Indomethacin (35 μg, Sigma, catalog #17378) or vehicle control(PBS) was injected into the TA muscle. The contralateral TA was used asan internal control, except for the force measurement experiments whereeach mouse had both legs injured with the same condition and independentmice were used for each condition.

For Pax7^(CreERT2);Rosa26-LSL-Luc mice experiments, we treated mice withfive consecutive daily intraperitoneal injections of tamoxifen toactivate luciferase expression under the control of the Pax7 promoter. Aweek after the last tamoxifen injection, mice were subjected tointramuscular injection of 10 μl of cardiotoxin (10 μM; Latoxan), whichwe designated as day 0 of the assay. Three days later either 13 nmoldmPGE2 or vehicle control (PBS) was injected into the TA muscle. Thecontralateral TA was used as an internal control. Bioluminescence wasassayed at days 3, 7, 10 and 14 post-injury.

For Pax7^(CreERT2); Ep4^(flox/flox) mice experiments, we treated micewith five consecutive daily intraperitoneal injections of tamoxifen toexcise the EP4 allele in Pax7 expressing cells. A week after the lasttamoxifen injection, mice were subjected to intramuscular injection of10 μl of notexin (10 μg ml⁻¹; Latoxan), which we designated as day 0 ofthe assay. As control mice, Pax7^(+/+); EP4^(flox/+) littermates of thesame sex were used.

We isolated and enriched muscle stem cells as previously described (24,26, 27). Briefly, mouse hind-limb muscles were isolated and dissociatedusing the gentleMACS Octo Dissociator with a modified manufacturerprotocol (Miltenyi Biotech). Dissociated muscle was digested with 0.2%collagenase (Roche) for 60 min, followed by collagenase/dispase (0.04 Uml⁻¹; Roche) digestion for 30 minutes. Mononucleated cells wereliberated by syringe dissociation with an 18 G needle. For mouse musclestem cells, single cell suspension were incubated with biotinylatedantibodies against CD11 b (1:800), CD45 (1:500), Scal (1:200) and CD31(1:200), followed by incubation with streptavidin magnetic beads(Miltenyi Biotech), streptavidin-APC-Cy7, integrin-α₇-PE (1:500) andCD34-eFluor660 (1:67). The cell mixture was depleted for hematopoieticlineage expressing and non-muscle cells on a magnetic based selectioncolumn (Miltenyi) for biotin-positive cells. The remaining cell mixturewas then subjected to FACS analysis to sort forCD45⁻CD11b⁻CD31⁻Scal⁻CD34⁺integrin-α₇ ⁺ MuSCs with >95% purity(DIVA-Van, Becton-Dickinson). We generated and analyzed flow cytometryscatter plots using FlowJo v10.0. For wild-type MuSC sorts, we pooledtogether MuSCs (˜5,000 each) from at least three independent age- andsex-matched donor mice.

We analyzed NURR1 levels by flow cytometry using myogenic progenitorsafter a 24 hr treatment of vehicle (DMSO) or PGE2 (10 ng/ml), or fromMuSCs transfected with shSCR or shNurr1 (see Knockdown assays section).We collected cells by incubation with 0.5% trypsin in PBS for 2 min at37° C. We fixed the cells using 1.6% paraformaldehyde in PBS and thenpermeabilized them in ice-cold methanol. We then blocked them instaining buffer (0.5% BSA in PBS) for 30 min at room temperature andstained them with a Mouse monoclonal anti-Nurr1 (Abcam, catalog#ab41917, 1:75) primary antibody or anti-mouse IgG control (JacksonImmunoResearch Laboratories). Then, we stained cells with PacificBlue-conjugated goat anti-mouse secondary antibody (Thermo FisherScientific, catalog #P-10994, 1:500). We analyzed the cells on a FACSLSR II cytometer using FACSDiva software (BD Biosciences) in theStanford Shared FACS Facility, purchased using an NIH S10 SharedInstrument Grant (S10RR027431-01).

We transplanted 250 MuSCs (FIG. 13A) or 1,000 MuSCs (FIG. 14E) from cellculture directly into the TA muscles of recipient mice as previouslydescribed (24, 26, 27). For wild-type MuSC studies, we transplantedcells from GFP/luc mice (2-4 mo of age) into hindlimb-irradiated NSGmice. For EP4^(flox/flox) MuSCs studies, we transplanted cells fromEP4^(flox/flox) mice (2-4 mo) that were transduced with a luc-IRES-GFPlentivirus (GFP/luc virus) and a subgroup received either a mCherry/Crelentivirus or a mock infection on day 2 of culture for a period of 24 hrbefore transplantation, as previously described (26) (see below “Musclestem cell culture, treatment and lentiviral infection” section fordetails). Prior to transplantation of muscle stem cells, we anesthetizedNSG recipient mice with ketamine (2.4 mg per mouse) by intraperitonealinjection. We then irradiated hindlimbs with a single 18 Gy dose, withthe rest of the body shielded in a lead jig. We performedtransplantations within 2 days of irradiation. We resuspended MuSCs atdesired cell concentrations in 0.1% gelatin/PBS and then transplantedthem (250 or 100 mouse MuSCs per TA) by intramuscular injection into theTA muscles in a 15 μl volume. For fresh MuSCs transplantation, wecoinjected sorted cells with 13 nmol of 16,16-Dimethyl Prostaglandin E2(dmPGE2) (Tocris, catalog #4027) or vehicle control (PBS). One monthafter transplant, we injected 10 μl of notexin (10 μg ml−1; Latoxan,France) to injure recipient muscles and to re-activate MuSCs in vivo. Wecompared cells from different conditions by transplantation into the TAmuscles of contralateral legs in the same mice. Three or eight weeksafter transplantation as indicated in the figure legends, mice wereeuthanized and the TAs were collected for analysis.

We performed bioluminescence imaging (BLI) using a Xenogen-100 system,as previously described (24, 26, 27, 30). Briefly, we anesthetized miceusing isofluorane inhalation and administered 120 μL D-luciferin (0.1mmol kg⁻¹, reconstituted in PBS; Caliper LifeSciences) byintraperitoneal injection. We acquired BLI using a 60s exposure atF-stop=1.0 at 5 minutes after luciferin injection. Digital images wererecorded and analyzed using Living Image software (CaliperLifeSciences). We analyzed images with a consistent region-of-interest(ROI) placed over each hindlimb to calculate a bioluminescence signal.We calculated a bioluminescence signal in radiance (p s⁻¹ cm⁻² sr⁻¹)value of 10⁴ to define an engraftment threshold. This radiance thresholdof 10⁴ is approximately equivalent to the total flux threshold of 10⁵p/s defined by the region of interest of similar size as reportedpreviously. This BLI threshold corresponds to the histological detectionof one or more GFP+ myofibers (24, 26, 27). We performed BLI imagingevery week after transplantation.

We fabricated polyethylene glycol (PEG) hydrogels from PEG precursors,synthesized as described previously (27). Briefly, we produced hydrogelsby using the published formulation to achieve 12-kPa (Young's modulus)stiffness hydrogels in 1 mm thickness, which is the optimal conditionfor culturing MuSCs and maintaining stem cell fate in culture (27). Wefabricated hydrogel microwell arrays of 12-kPa for clonal proliferationexperiments, as described previously (27). We cut and adhered allhydrogels to cover the surface area of 12-well or 24-well cultureplates.

Following isolation, we resuspended MuSCs in myogenic cell culturemedium containing DMEM/F10 (50:50), 20% FBS, 2.5 ng ml⁻¹ lfibroblastgrowth factor-2 (FGF-2 also known as bFGF) and 1%penicillin-streptomycin. We seeded MuSC suspensions at a density of 500cells per cm² surface area. We maintained cell cultures at 37° C. in 5%CO₂ and changed medium every other day. For PGE2, EP4 receptorantagonist treatment studies, we added 1-200 ng/ml Prostaglandin E2(Cayman Chemical) (unless specified in the figure legends, 10 ng/ml wasthe standard concentration used), and/or 1 μM EP4 antagonist(ONO-AE3-208, Cayman Chemical), to the MuSCs cultured on collagen coateddishes for the first 24 h. The cells were then trypsinized and cellsreseeded onto hydrogels for an additional 6 days of culture. Alltreatments were compared to their solvent (DMSO) vehicle control. Forstripped serum assays, we resuspended isolated MuSCs in stripped serummedium containing DMEM/F10 (50:50), 20% charcoal stripped FBS (Gibco,cat #12676011), 2.5 ng ml⁻¹ bFGF and 1% penicillin-streptomycin. Forthese experiments MuSCs were cultured on hydrogels and vehicle (DMSO) or10 ng/ml PGE2 (Cayman Chemical) was added to the cultures with everymedia change (every two days). Proliferation (see below) was assayed 7days later. We performed all MuSC culture assays and transplantationsafter 1 week of culture unless noted otherwise.

For EP4^(f/f) MuSCs studies, we isolated MuSCs as described above(Muscle stem cell isolation), and infected all cells with lentivirusencoding EF1α-luc-IRES-GFP (GFP/luc virus) for 24 h in culture asdescribed previously (26) and a subset of them was coinfected with alentivirus encoding pLM-CMV-R-Cre (mCherry/Cre virus) for 24 h inculture. pLM-CMV-R-Cre was a gift from Michel Sadelain (Addgene plasmid#27546) (50). We transplanted EP4^(f/f) MuSCs (1,000 cells) into young(2-4 mo) 18-gy irradiated TAs of NSG recipient mice. For in vitroproliferation assays, EP4^(f/f) MuSCs were plated on hydrogelspost-infection and treated for 24 hr with vehicle (DMSO) or 10 ng/mlPGE2, and proliferation was assayed 3 days later. Cells were assayed forGFP and/or mCherry expression 48 h post-infection using an invertedfluorescence microscope (Carl Zeiss Microimaging). Additionally, we alsoperformed experiments with MuSCs isolated from Pax7^(CreERT2);EP4^(flox/flox) or control Pax7^(+/+); EP4^(flox/+) littermates. MuSCswere plated on collagen-coated plates and treated with 1 μM 4-hydroxytamoxifen or vehicle (95% Ethanol) during 48 h and then either passedonto hydrogels to assess proliferation 7 days later or treated with PGE2or vehicle and collected for analysis. MuSCs are freshly isolated fromthe mice by FACS and put in culture for a maximum time period of oneweek, therefore mycoplasma contamination is not assessed.

We assayed clonal muscle stem cell proliferation by time-lapsemicroscopy as previously described (26, 27). Briefly, we sorted MuSCsfrom C57Bl/6 mice (2-4 months), plated them on collagen-coated platesand treated them PGE2 (Cayman Chemical) or vehicle (DMSO) for 24 hr.Cells were then trypsinized and reseeded at a density of 500 cells percm² surface area in hydrogel microwells with 600 μm diameter. Fortime-lapse microscopy we monitored cell proliferation for those wellswith single cells for 38 h days after seeding and recorded images every3 min at 10× magnification using a PALM/AxioObserver Z1 system (CarlZeiss MicroImaging) with a custom environmental control chamber andmotorized stage. We analyzed time-lapse image sequences using the BaxterAlgorithms for Cell Tracking and Lineage Reconstruction to identify andtrack single cells and generate lineage trees (26-28, 51, 52).

Viable and dead cells were distinguished in time-lapse sequences basedon phase-contrast boundary and motility maintenance or loss,respectively. The proportion of live cells in each generation (G1-G6) atall timepoints is shown as cell number normalized to a startingpopulation of 100 single MuSCs. The data analysis was blinded. Theresearchers performing the imaging acquisition and scoring were unawareof the treatment condition given to sample groups analyzed.

To assay proliferation, we used three different assays (hemocytometer,VisionBlue, and EdU). For each, we seeded MuSCs on flat hydrogels(hemocytometer and VisionBlue) or collagen-coated plates (EdU assay) ata density of 500 cells per cm² surface area. For hemocytometer cellnumber count, we collected cells at indicated timepoints by incubationwith 0.5% trypsin in PBS for 5 min at 37° C. and quantified them using ahemocytometer at least 3 times. Additionally, we used the VisionBlueQuick Cell Viability Fluorometric Assay Kit (BioVision, catalog #K303)as a readout for cell growth in culture. Briefly, we incubated MuSCswith 10% VisionBlue in culture medium for 3 h, and measured fluorescenceintensity on a fluorescence plate reader (Infinite M1000 PRO, Tecan) atEx=530-570 nm, Em=590-620 nm. We assayed proliferation using theClick-iT EdU Alexa Fluor 555 Imaging kit (Life Technologies). Briefly,we incubated live cells with EdU (20 μM) for 1 hr prior to fixation, andstained nuclei according to the manufacturer's guidelines together withanti-MYOGENIN (Santa Cruz, catalog #sc576, 1:250) to assaydifferentiation. We counterstained nuclei with DAPI (Invitrogen). Weacquired images with an AxioPlan2 epifluorescent microscope (Carl ZeissMicroimaging) with Plan NeoFluar 10×/0.30NA or 20×/0.75NA objectives(Carl Zeiss) and an ORCA-ER digital camera (Hamamatsu Photonics)controlled by SlideBook (3i) software. We quantified EdU positive cellsusing the MetaMorph Image Analysis software (Molecular Devices). Dataanalyses were blinded, where researchers performing cell scoring wereunaware of the treatment condition given to sample groups analyzed.

For Nurr1 silencing in MuSCs, lentiviruses containing pLKO.1-scrambleshRNA (shSCR) and pLKO.1-Nurr1 shRNA (Mission shRNA, TRCN0000026029,Sigma) were produced in 293T cells using the packaging plasmids pLP1,pLP2 and pLP/VSVG (Invitrogen), by cotransfecting all plasmids usingFuGENE-6 (Promega) according to the manufacturer's protocol. Cells wereplated the day prior to infection, and supernatants were collected every12 hours for two days from 293T cells. Freshly sorted MuSCs were seededon collagen-coated plates for 24 hrs and were then infected with thelentiviruses. 48 hrs after, cells were passed onto hydrogels and treatedwith PGE2 or vehicle (DMSO) for 24 hrs. Proliferation was assayed 7 dayslater.

We isolated RNA from MuSCs using the RNeasy Micro Kit (Qiagen). Formuscle samples, we snap froze the tissue in liquid nitrogen, homogenizedthe tissues using a mortar and pestle, followed by syringe and needletrituration, and then isolated RNA using Trizol (Invitrogen). Wereverse-transcribed cDNA from total mRNA from each sample using theSensiFAST™ cDNA Synthesis Kit (Bioline). We subjected cDNA to RT-PCRusing SYBR Green PCR Master Mix (Applied Biosystems) or TaqMan Assays(Applied Biosystems) in an ABI 7900HT Real-Time PCR System (AppliedBiosystems). We amplified samples at 95° C. for 10 min and then 40cycles at 95° C. for 15 s and 60° C. for 1 min. To quantify relativetranscript levels, we used 2^(-ΔΔCt) to compare treated and untreatedsamples and expressed the results relative to Gapdh. For SYBR GreenqRT-PCR, we used the following primer sequences: Gapdh, forward5′-TTCACCACCATGGAGAAGGC-3′, reverse 5′-CCCTTTTGGCTCCACCCT-3; Ptges,forward 5′-GCTGTCATCACAGGCCAGA-3′, reverse 5′-CTCCACATCTGGGTCACTCC-3;Ptges2, forward 5′-CTCCTACAGGAAAGTGCCCA-3′, reverse5′-ACCAGGTAGGTCTTGAGGGC-3′; Ptger1, forward 5′ GTGGTGTCGTGCATCTGCT-3′,reverse, 5′ CCGCTGCAGGGAGTTAGAGT-3′, and Ptger2, forward5′-ACCTTCGCCATATGCTCCTT-3′, reverse 5′-GGACCGGTGGCCTAAGTATG-3′. TaqManAssays (Applied Biosystems) were used to quantify Pax7, Myogenin, Nurr1, Ptger3 and Ptger4 in samples according to the manufacturerinstructions with the TaqMan Universal PCR Master Mix reagent kit(Applied Biosystems). Transcript levels were expressed relative to Gapdhlevels. For SYBR Green qPCR, Gapdh qPCR was used to normalize input cDNAsamples. For Taqman qPCR, multiplex qPCR enabled target signals (FAM) tobe normalized individually by their internal Gapdh signals (VIC).

Muscle was harvested, rinsed in ice-cold PBS containing indomethacin(5.6 μg/ml), and snap frozen in liquid nitrogen. Frozen samples werepulverized in liquid nitrogen. The powder was transferred to anEppendorf tube with 500 μl of lysate buffer (50 mM Tris-HCl pH 7.5, 150mM NaCl, 4 mM CaCl, 1.5% Triton X-100, protease inhibitors andmicrococcal nuclease), and then homogenized using a tissue homogenizer.The PGE2 level of the supernatant was measured using a PGE2 ELISA Kit(R&D Systems, catalog #KGE004B) and expressed relative to total proteinmeasured by BCA assay (BioRad) and expressed as ng of PGE2. Each samplewas assayed in duplicate and in each of two independent experiments.

For conditioned medium assays, muscle fibers from the extensor digitorumlongus (EDL) were isolated as previously described (53). Fibers werecultured in stripped serum medium in the presence or absence ofindomethacin (1 μM, Sigma) for 24 hours. Conditioned medium wascollected and measured using the PGE2 ELISA Kit (R&D Systems, catalog#KGE004B) and expressed relative to the collected volume (ml). Eachsample was assayed in triplicate and in two independent experiments.

MuSCs were treated with DMSO (vehicle) or PGE2 (10 ng/ml) for 1 h andcyclic AMP levels measured according to the cAMP-Glo Assay protocoloptimized by the manufacturer (Promega). Each sample was assayed intriplicate and in two independent experiments.

Mice were injured as described in the Muscle injury section. Forcemeasurements were on the TA muscles at day 14 post-injury based onprotocols published previously (26, 54). Briefly, mice were anesthetizedwith 2-5% vaporized Isoflurane mixed with O₂. Mice were positioned undera heat lamp in order to maintain the body and muscle temperature at 30°C. The distal tendon of the TA muscle was dissected and tied to a300C-LR force transducer (Aurora Scientific) by surgical suture. Kneesof the animals were secured to a fixed steel post and their feet weretaped to the platform to prevent movement from the contraction of othermuscle groups. Electrical stimulations were applied across two needleelectrodes, placed through the skin just above the knee and beneath theTA muscle to stimulate the tibial nerve. In all measurements, we used0.1-ms pulses at a predetermined supramaximal stimulation voltage. TAmuscles were stimulated with a single 0.1-ms pulse for twitch forcemeasurements, and a train of 150 Hz for 0.3 s pulses for tetanic forcemeasurements. We performed five twitch and then five tetanicmeasurements on each muscle, with 2-3 min recovery between eachmeasurement. Data were collected with a PCI-6251 acquisition card(National Instruments) and analyzed in Matlab. We calculated specificforce values by normalizing the force measurements by the musclephysiological cross-sectional areas (PCSAs), which were similar betweengroups. PCSA (measured in mm²) was calculated according to the followingequation (55):

PCSA (mm²)=[mass (g)×Cos θ]±[ρ(g/mm³)×fiber length (mm)],

where θ is pennation angle of the fiber and ρ is muscle density(0.001056 g/mm³).

For RNA sequencing, α7-integrin⁺CD34⁺ muscle stem cells were isolated asdescribed above, seeded on collagen-coated plates, treated a day laterwith PGE2 or vehicle (DMSO) and processed after 24 hours of treatment.RNA was isolated using Qiagen RNAEasy Micro kit from 5,000-10,000 cells,and cDNA generated and amplified using NuGEN Ovation RNA-Seq System v2kit. Libraries were constructed from cDNA with the TruSEQ RNA LibraryPreparation Kit v2 (Illumina), and sequenced to 30-40×106 1×75 bp readsper sample on a HiSEQ 2500 from the Stanford Functional GenomicsFacility, purchased using an NIH S10 Shared Instrument Grant(S10OD018220).

For the RNA-Seq analysis, RNA sequences were aligned against the Musmusculus genome using STAR (56). RSEM (57) was used for callingtranscripts and calculating transcripts per million (TPM) values, aswell as total counts. A counts matrix containing the number of countsfor each gene and each sample was obtained. This matrix was analyzed byDESeq to calculate statistical analysis of significance (58) of genesbetween samples.

We collected and prepared recipient TA muscle tissues for histology aspreviously described (26, 27). For mouse injury assays we incubatedtransverse sections with Rabbit polyclonal anti-PGE2 (abcam, Catalog#ab2318, 1:100) Rat polyclonal anti-Laminin (Clone A5) (EMD Millipore,Catalog #05-206, 1:200), Mouse monoclonal anti-Pax7 (Santa Cruz, Catalog#sc-81648, 1:50), AlexaFluor 647-conjugated wheat germ agglutinin (WGA)antibody (W32466, Thermo Fisher Scientific), Rabbit polyclonal anti-GFP(A11122, Thermo Fisher Scientific, 1:500) and Mouse monoclonalanti-Embryonic Myosin Heavy Chain (DSHB, Catalog #F1.652, 1:10) primaryantibodies and then with AlexaFluor secondary Antibodies (JacksonImmunoResearch Laboratories, 1:500). We counterstained nuclei with DAPI(Invitrogen).

We acquired images with an AxioPlan2 epifluorescent microscope (CarlZeiss Microimaging) with Plan NeoFluar 10×/0.30NA or 20×/0.75NAobjectives (Carl Zeiss) and an ORCA-ER digital camera (HamamatsuPhotonics) controlled by the SlideBook (3i) software or with a KEYENCEBZ-X700 all-in-one fluorescence microscope (Keyence, Osaka, Japan) witha 20×/0.75NA objectives. The images were cropped using Adobe Photoshopwith consistent contrast adjustments across all images from the sameexperiment. The image composites were generated using Adobe Illustrator.We analyzed the number of PAX7 positive cells using the MetaMorph ImageAnalysis software (Molecular Devices), and the fiber area using theBaxter Algorithms for Myofiber Analysis that identified the fibers andsegmented the fibers in the image to analyze the area of each fiber. ForPAX7 quantification we examined serial sections spanning a depth of atleast 2mm of the TA. For fiber area the entire cross-section of the TAwith the largest injured area was captured and stitched using theKeyence Analysis Software. Data capture and analyses were blinded. Theresearchers performing the imaging acquisition and scoring were unawareof treatment condition given to sample groups analyzed.

We performed cell culture experiments in at least three independentexperiments where three biological replicates were pooled in each. Ingeneral, we performed MuSC transplant experiments in at least twoindependent experiments, with at least 3-5 total transplants percondition. We used a paired t-test for experiments where control sampleswere from the same experiment in vitro or from contralateral limbmuscles in vivo. A non-parametric Mann-Whitney test was used todetermine the significance difference between vehicle-treated vsPGE2-treated groups using α=0.05. ANOVA or multiple t-test was performedfor multiple comparisons with significance level determined usingBonferroni correction or Fisher's test as indicated in the figurelegends. Unless otherwise described, data are shown as the mean±s.e.m.Differences with p value<0.05 were considered significant (*p<0.05,**p<0.01, ***p<0.001, ****p<0.0001).

RNASeq data have been submitted in MIME-compliant format to GEO,accession number GSE97375.

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Muscle injury: We used an injury model entailing intramuscular injectionof 10 μl of notexin (10 μg ml⁻¹; Latoxan, catalog #L8104) or cardiotoxin(10 μM; Latoxan, catalog #L8102) into the Tibilais anterior (TA) muscle.For cryoinjury, an incision was made in the skin overlying the TA muscleand a copper probe, chilled in liquid nitrogen, was applied to the TAmuscle for three 10 s intervals, allowing the muscle to thaw betweeneach application of the cryoprobe. When indicated, 48 hr after injuryeither 16,16-Dimethyl Prostaglandin E2 (dmPGE2) (13 nmol, Tocris,catalog #4027), Indomethacin (35 μg, Sigma, catalog #17378) or vehiclecontrol (PBS) was injected into the TA muscle. The contralateral TA wasused as an internal control, except for the force measurementexperiments where each mouse had both legs injured with the samecondition and independent mice were used for each condition.

For Pax7^(CreERT2);Rosa26-LSL-Luc mice experiments, we treated mice withfive consecutive daily intraperitoneal injections of tamoxifen toactivate luciferase expression under the control of the Pax7 promoter. Aweek after the last tamoxifen injection, mice were subjected tointramuscular injection of 10 μl of cardiotoxin (10 μM; Latoxan), whichwe designated as day 0 of the assay. Three days later either 13 nmoldmPGE2 or vehicle control (PBS) was injected into the TA muscle. Thecontralateral TA was used as an internal control. Bioluminescence wasassayed at days 3, 7, 10 and 14 post-injury.

For Pax7^(CreERT2); Ep4^(flox/flox) mice experiments, we treated micewith five consecutive daily intraperitoneal injections of tamoxifen toexcise the EP4 allele in Pax7 expressing cells. A week after the lasttamoxifen injection, mice were subjected to intramuscular injection of10 μl of notexin (10 μg ml⁻¹; Latoxan), which we designated as day 0 ofthe assay. As control mice, Pax7^(+/+); EP4^(flox/+) littermates of thesame sex were used.

Fluorescence activated cell sorting for mouse muscle stem cells: Formuscle stem cell isolation, mouse hind-limb muscles were dissociatedusing the gentleMACS Octo Dissociator with a modified manufacturerprotocol (Miltenyi Biotech). Dissociated muscle was digested with 0.2%collagenase (Roche) for 60 min, followed by collagenase/dispase (0.04 Uml⁻¹; Roche) digestion for 30 minutes. Mononucleated cells wereliberated by syringe dissociation with an 18 G needle. For mouse musclestem cells, single cell suspension were incubated with biotinylatedantibodies against CD11 b (1:800), CD45 (1:500), Scal (1:200) and CD31(1:200), followed by incubation with streptavidin magnetic beads(Miltenyi Biotech), streptavidin-APC-Cy7, integrin-α₇-PE (1:500) andCD34-eFluor660 (1:67). The cell mixture was depleted for hematopoieticlineage expressing and non-muscle cells on a magnetic based selectioncolumn (Miltenyi) for biotin-positive cells. The remaining cell mixturewas then subjected to FACS analysis to sort forCD45⁻CD11b⁻CD31⁻Scal⁻CD34+integrin-α₇ ⁺ MuSCs with >95% purity(DIVA-Van, Becton-Dickinson). We generated and analyzed flow cytometryscatter plots using FlowJo v10.0. For wild-type MuSC sorts, we pooledtogether MuSCs (˜5,000 each) from at least three independent age- andsex-matched donor mice.

Muscle stem cell transplantation: For wild-type MuSC studies, wetransplanted cells from GFP/luc mice (2-4 mo of age) intohindlimb-irradiated NSG mice. For EP4^(flox/flox) MuSCs studies, wetransplanted cells from EP4^(flox/flox) mice (2-4 mo) that weretransduced with a luc-IRES-GFP lentivirus (GFP/luc virus) and a subgroupreceived either a mCherry/Cre lentivirus or a mock infection on day 2 ofculture for a period of 24 hr before transplantation, as previouslydescribed (26) (see “Muscle stem cell culture, treatment and lentiviralinfection” section for details). Prior to transplantation of muscle stemcells, we anesthetized NSG recipient mice with ketamine (2.4 mg permouse) by intraperitoneal injection. We then irradiated hindlimbs with asingle 18 Gy dose, with the rest of the body shielded in a lead jig. Weperformed transplantations within 2 days of irradiation. We resuspendedMuSCs at desired cell concentrations in 0.1% gelatin/PBS and thentransplanted them (250 or 100 mouse MuSCs per TA) by intramuscularinjection into the TA muscles in a 15 μl volume. For fresh MuSCstransplantation, we coinjected sorted cells with 13 nmol of16,16-Dimethyl Prostaglandin E2 (dmPGE2) (Tocris, catalog #4027) orvehicle control (PBS). One month after transplant, we injected 10 μl ofnotexin (10 μg ml−1; Latoxan, France) to injure recipient muscles and tore-activate MuSCs in vivo. We compared cells from different conditionsby transplantation into the TA muscles of contralateral legs in the samemice. Three or eight weeks after transplantation as indicated in thefigure legends, mice were euthanized and the TAs were collected foranalysis.

Bioluminescent Imaging: For bioluminescence imaging (BLI), weanesthetized mice using isofluorane inhalation and administered 120 μLD-luciferin (0.1 mmol kg⁻¹, reconstituted in PBS; Caliper LifeSciences)by intraperitoneal injection. We acquired BLI using a 60 s exposure atF-stop=1.0 at 5 minutes after luciferin injection. Digital images wererecorded and analyzed using Living Image software (CaliperLifeSciences). We analyzed images with a consistent region-of-interest(ROI) placed over each hindlimb to calculate a bioluminescence signal.We calculated a bioluminescence signal in radiance (p s⁻¹ cm⁻² sr⁻¹)value of 10⁴ to define an engraftment threshold. This radiance thresholdof 10⁴ is approximately equivalent to the total flux threshold of 10⁵p/s defined by the region of interest of similar size as reportedpreviously. This BLI threshold corresponds to the histological detectionof one or more GFP+ myofibers (24, 26, 27). We performed BLI imagingevery week after transplantation.

Hydrogel fabrication: We produced hydrogels by using the publishedformulation to achieve 12-kPa (Young's modulus) stiffness hydrogels in 1mm thickness, which is the optimal condition for culturing MuSCs andmaintaining stem cell fate in culture (27). We fabricated hydrogelmicrowell arrays of 12-kPa for clonal proliferation experiments, asdescribed previously (27). We cut and adhered all hydrogels to cover thesurface area of 12-well or 24-well culture plates.

Clonal muscle stem cell proliferation and fate analyses: To perform timelapse-analysis, we sorted MuSCs from C57Bl/6 mice (2-4 months), platedthem on collagen-coated plates and treated them PGE2 (Cayman Chemical)or vehicle (DMSO) for 24 hr. Cells were then trypsinized and reseeded ata density of 500 cells per cm² surface area in hydrogel microwells with600 μm diameter. For time-lapse microscopy we monitored cellproliferation for those wells with single cells for 38 h days afterseeding and recorded images every 3 min at 10× magnification using aPALM/AxioObserver Z1 system (Carl Zeiss MicroImaging) with a customenvironmental control chamber and motorized stage. We analyzedtime-lapse image sequences using the Baxter Algorithms for Cell Trackingand Lineage Reconstruction to identify and track single cells andgenerate lineage trees (26-28, 51, 52).

Viable and dead cells were distinguished in time-lapse sequences basedon phase-contrast boundary and motility maintenance or loss,respectively. The proportion of live cells in each generation (G1-G6) atall timepoints is shown as cell number normalized to a startingpopulation of 100 single MuSCs. The data analysis was blinded. Theresearchers performing the imaging acquisition and scoring were unawareof the treatment condition given to sample groups analyzed.

Proliferation assays: To assay proliferation, we used three differentassays (hemocytometer, VisionBlue, and EdU). For each, we seeded MuSCson flat hydrogels (hemocytometer and VisionBlue) or collagen-coatedplates (EdU assay) at a density of 500 cells per cm² surface area. Forhemocytometer cell number count, we collected cells at indicatedtimepoints by incubation with 0.5% trypsin in PBS for 5 min at 37° C.and quantified them using a hemocytometer at least 3 times.Additionally, we used the VisionBlue Quick Cell Viability FluorometricAssay Kit (BioVision, catalog #K303) as a readout for cell growth inculture. Briefly, we incubated MuSCs with 10% VisionBlue in culturemedium for 3 h, and measured fluorescence intensity on a fluorescenceplate reader (Infinite M1000 PRO, Tecan) at Ex=530-570 nm, Em=590-620nm. We assayed proliferation using the Click-iT EdU Alexa Fluor 555Imaging kit (Life Technologies). Briefly, we incubated live cells withEdU (20 μM) for 1 hr prior to fixation, and stained nuclei according tothe manufacturer's guidelines together with anti-MYOGENIN (Santa Cruz,catalog #sc576, 1:250) to assay differentiation. We counterstainednuclei with DAPI (Invitrogen). We acquired images with an AxioPlan2epifluorescent microscope (Carl Zeiss Microimaging) with Plan NeoFluar10×/0.30NA or 20×/0.75NA objectives (Carl Zeiss) and an ORCA-ER digitalcamera (Hamamatsu Photonics) controlled by SlideBook (3i) software. Wequantified EdU positive cells using the MetaMorph Image Analysissoftware (Molecular Devices). Data analyses were blinded, whereresearchers performing cell scoring were unaware of the treatmentcondition given to sample groups analyzed.

PGE2 ELISA: Muscle was harvested, rinsed in ice-cold PBS containingindomethacin (5.6 μg/ml), and snap frozen in liquid nitrogen. Frozensamples were pulverized in liquid nitrogen. The powder was transferredto an Eppendorf tube with 500 μl of lysate buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 4 mM CaCl, 1.5% Triton X-100, protease inhibitors andmicrococcal nuclease), and then homogenized using a tissue homogenizer.The PGE2 level of the supernatant was measured using a PGE2 ELISA Kit(R&D Systems, catalog #KGE004B) and expressed relative to total proteinmeasured by BCA assay (BioRad) and expressed as ng of PGE2. Each samplewas assayed in duplicate and in each of two independent experiments.

For conditioned medium assays, muscle fibers from the extensor digitorumlongus (EDL) were isolated as previously described (53). Fibers werecultured in stripped serum medium in the presence or absence ofindomethacin (1 μM, Sigma) for 24 hours. Conditioned medium wascollected and measured using the PGE2 ELISA Kit (R&D Systems, catalog#KGE004B) and expressed relative to the collected volume (ml). Eachsample was assayed in triplicate and in two independent experiments.

In vivo muscle force measurement: To perform force measurments, micewere first anesthetized with 2-5% vaporized Isoflurane mixed with O₂.Mice were positioned under a heat lamp in order to maintain the body andmuscle temperature at 30° C. The distal tendon of the TA muscle wasdissected and tied to a 300C-LR force transducer (Aurora Scientific) bysurgical suture. Knees of the animals were secured to a fixed steel postand their feet were taped to the platform to prevent movement from thecontraction of other muscle groups. Electrical stimulations were appliedacross two needle electrodes, placed through the skin just above theknee and beneath the TA muscle to stimulate the tibial nerve. In allmeasurements, we used 0.1-ms pulses at a predetermined supramaximalstimulation voltage. TA muscles were stimulated with a single 0.1-mspulse for twitch force measurements, and a train of 150 Hz for 0.3 spulses for tetanic force measurements. We performed five twitch and thenfive tetanic measurements on each muscle, with 2-3 min recovery betweeneach measurement. Data were collected with a PCI-6251 acquisition card(National Instruments) and analyzed in Matlab. We calculated specificforce values by normalizing the force measurements by the musclephysiological cross-sectional areas (PCSAs), which were similar betweengroups. PCSA (measured in mm²) was calculated according to the followingequation (55):

PCSA (mm²)=[mass (g)×Cos θ]±[ρ(g/mm³)×fiber length (mm)],

where θ is pennation angle of the fiber and ρ is muscle density(0.001056 g/mm³).

Example 4 Study to Establish an Optimal PGE2 Dosage for Use inCombination with Bupivacaine and Monitor MuSC Numbers and MuscleRegeneration in Mice

This example describes the use of a double-transgenic mouse model andbioluminescent imaging to examine the effects of compositions of thepresent invention on muscle regeneration and to establish an optimaldose of PGE2 or a PGE2 derivative (16,16-dimethyl prostaglandin E2) foruse in conjunction with bupivacaine.

Double-transgenic Pax7^(creERT2);Rosa26-LSL-Luc mice were generated bycrossing Pax7^(CreERT2) mice and Rosa26-LSL-Luc mice obtained fromJackson Laboratory (Stock #005125). These genotypes were validated byappropriate PCR-based strategies. All mice from transgenic and wild-typestrains were of young age (2-4 months). All experiments were conductedusing age and gender-matched controls. As depicted in the experimentalscheme shown in FIG. 23A, mice were treated with five consecutive dailyintraperitoneal injections of tamoxifen to activate luciferaseexpression under the control of the Pax7 promoter. A week after the lasttamoxifen injection, the tibialis anterior (TA) muscles were injectedintramuscularly with 50 μL of a drug mixture containing 0.125%, 0.25%,or 0.5% bupivacaine (BPV) (Cayman Chemical Cat #16618) and 10 μg16,16-dimethyl prostaglandin E2 (dmPGE2; Tocris, Catalog #4027) using a30 gauge needle on a Hamilton syringe. The contralateral TA received 50μL of a mixture containing 0.5% bupivacaine (BPV) and DMSO vehicle asbaseline sham control. Bioluminescence was assayed at days 3, 7, 10 and14 post-injury.

Bioluminescence imaging (BLI) was performed using a Xenogen-100 system,as previously described. Briefly, mice were anesthetized usingisofluorane inhalation and administered 120 μL D-luciferin (0.1 mmol/kg,reconstituted in PBS; Caliper LifeSciences) by intraperitonealinjection. BLI was acquired with between 5-60 second exposure atF-stop=1.0 at 5 minutes after luciferin injection. Digital images wererecorded and analyzed using Living Image software (CaliperLifeSciences). Images were analyzed with a consistent region-of-interest(ROI) placed over each hindlimb to calculate a bioluminescence signal. Abioluminescence signal was calculated in radiance (p s⁻¹ cm⁻² sr⁻¹)value of 10⁴ to define a positive threshold signal over background. BLIimaging was performed bi-weekly for 2 weeks after coinjection of BPVwith dmPGE2 or vehicle.

The data in FIGS. 23A-C and 24 are shown as the mean±s.e.m. with n=6mice. Multiple t-test for each time point or one-way ANOVA was performedto compare between treatment (BPV/dmPGE2) and control (BPV/vehicle)group. Differences with p value<0.05, denoted as * (asterisk), wereconsidered significant.

FIG. 23B shows examples of BLI signals, which were higher in musclesthat were treated with a combination of BPV and dmPGE2 than muscles thatwere treated with BPV alone. The BLI images were obtained two weeksafter injection. As shown in FIG. 23C, the log fold change in BLI twoweeks after injection was significantly higher (p<0.05) in theBPV/dmPGE2 group compared to the BPV/vehicle group, indicating thatmuscle regeneration was more pronounced when a combination of BPV anddmPGE2 was used. No detectable change was noted in the negative control(DMSO vehicle only) and dmPGE2 only groups.

As shown in FIG. 24, when given in combination with dmPGE2, BPV produceda dose-dependent increase in muscle regeneration (as evidenced by largerfold changes in BLI signals), with statistically significant changesapparent two weeks after injection.

Together, these data show that a combination of dmPGE2 and BPV is moreeffective at promoting muscle regeneration than either dmPGE2 or BPValone, and that higher doses of dmPGE2 result in more pronounced muscleregeneration.

References: Safran, M. et al. Mouse reporter strain for noninvasivebioluminescent imaging of cells that have undergone Cre-mediatedrecombination. Molecular imaging 2, 297-302 (2003); Cosgrove, B. D. etal. Rejuvenation of the muscle stem cell population restores strength toinjured aged muscles. Nature medicine 20, 255-264 (2014); Gilbert, P. M.et al. Substrate elasticity regulates skeletal muscle stem cellself-renewal in culture. Science 329, 1078-1081 (2010); Sacco, A.,Doyonnas, R., Kraft, P., Vitorovic, S. & Blau, H. M. Self-renewal andexpansion of single transplanted muscle stem cells. Nature 456, 502-506(2008); Ho, A. T. & Blau, H. M. Noninvasive Tracking of Quiescent andActivated Muscle Stem Cell (MuSC) Engraftment Dynamics In Vivo. Methodsin molecular biology 1460, 181-189 (2016).

Example 5 Study to Establish an Optimal PGE2 Dosage for Use inCombination with Bupivacaine and Monitor MuSC Numbers and MuscleRegeneration in Mice

This example illustrates a study that can be performed to optimize theeffective dose of PGE2 together with bupivacaine. The bupivacainecomponent serves two purposes: it provides anesthesia for the injectionand also stimulates muscle stem cell function in regeneration. Thedosages to be tested comprise ranges that have been used previously inpatients for each drug alone. In each case, the total PGE2-bupivacainedosage is delivered in four injections in order to maximize muscle stemcell activation due to the needle and the mildly myotoxic anesthetic. Totest muscle regenerative capacity non-invasively by BLI, thePax7-Luciferase transgenic mouse model is used, which provides abioluminescence readout for increased endogenous stem cell functionnon-invasively over time. A model of sciatica nerve transection isemployed, which is a well-tolerated, validated, and reproducible modelof denervation-induced skeletal muscle atrophy in rodents. Thistechnique leads to a loss of muscle mass mimicking the atrophic abductorpollicis brevis (APB) muscles seen in patients with CTS. Then a bolus ofPGE2-bupivacaine is delivered into four randomized groups of mice totest the range of three PGE2 dosages that are compared to a controluntreated group. These experiments can be used to define the optimaldosage of an injected combination of PGE2 and bupivacaine for use in aclinical trial.

Both PGE2 and bupivacaine have previously been used for otherindications in human clinical trials (see, NCT01861665 and, but nottogether in muscle. For the studies described in this example, thepublished efficacious and FDA approved dose range that is at the NOAELof 0.5% Bupivacaine can be used. The purpose is to test for the optimalPGE2 dose that maximally augments endogenous murine MuSC function inatrophied hindlimb muscles. In these experiments, assessment ofendogenous MuSC expansion entails a series of rapid sensitive andnon-invasive BLI measurements over a time course. Finally, histology isespecially useful in analyses of the atrophied muscle cohort, todetermine the degree of denervation and early muscle damage responseinduced by a combination of PGET2 and bupivacaine injection andsubsequent muscle regeneration and re-innervation, which may beattributed to restoration of muscle functions at the treatment endpoint.

Establishing the Effective Dose of PGE2 in Stimulating Endogenous MuSCNumbers in Vivo

A bioluminescence imaging (BLI) assay is employed as a convenientnon-invasive method to quickly assess MuSC expansion in vivo, andprovide a sensitive measure of regeneration potential, by using a musclestem cell reporter mouse model (i.e., Pax7^(CreERT2); Rosa26-LSL-Luc)after inducing nerve transection induced atrophy and GA injection of acombination of PGE2 and bupivacaine. The PGE-bupivacaine total dosage ineach case is delivered in 4 injections to maximize exposure of theentire muscle and enhance muscle stem cell activation and regenerationdue to the needle and the mild myotoxin anesthetic.

BLI affords extraordinary sensitivity due to its high signal-to-noiseratio, as excitation light used in fluorescence imaging (which generatesbackground noise) is not required. The BLI signal emitted from theluciferin catalytic reaction by the luciferase expressed in the expandedendogenous muscle stem cells is directly recorded by a cooledcharge-coupled device (CCD) camera. This dynamic readout allowslongitudinal studies of MuSC functions in vivo to be conducted in thesame mouse over time, and therefore constitutes a useful adjunct toendstage serial sectioning and immunofluorescence analyses ofregenerated muscles.

Muscle regeneration is induced by using bupivacaine as a myotoxin.Bupivacaine has been found to be efficacious in muscleinjury/regeneration models in prior studies of muscle stem cells. Adosage of 0.5% bupivacaine, which is well documented to induce robustregeneration in mouse muscles, is used. This dose is comparable to thedose used clinically in patients for local anesthesia and peripheralnerve blocks.

A combined formulation of a fixed dose of bupivacaine is tested with arange of PGE2 concentrations, all of which are below the maximum FDAapproved dose of PGE2 (see, NCT00602095). An FDA-approved GMP-grade PGE2is used, which can be obtained, for example, from PfizerPharmaceuticals, UK.

The formulation comprising PGE2 and bupivacaine is delivered to theinduced atrophied gastrocnemius (GA) muscles of both legs, as the mixedfast and slow fiber type composition and size of the GA mouse muscles(135±22.6 mg) more closely approximates the human ABP muscle (261±119mg) than the tibialis anterior muscles studied previously. The mouse GAmuscle also has a similar fiber length/muscle length ratio (45.5%±4.5%)to the human APB muscle (69%±9%). Previous studies have shown thattransplanted MuSCs can restore strength to injured GA muscles ofdystrophic mice.

Mouse hindlimb muscle atrophy is induced using the established sciaticnerve transection model to mimic the atrophic APB muscles seen inpatients with CTS . This model causes a period of denervation of theposterior hindlimb muscles (i.e., after nerve transection) which resultsin atrophy, and is followed by reinnervation of the muscle (after thenerve axons have regenerated back to their motor endplates).

Using a MuSC reporter mouse model and the BLI technique, the dose mosteffective in enhancing endogenous MuSC regenerative function assayed byBLI is determined. Specifically, mice are treated with five consecutivedaily intraperitoneal injections of tamoxifen to activate luciferaseexpression. A week after the last tamoxifen injection, mice aresubjected to sciatic nerve transection. After two weeks, a time pointwhen muscle atrophy is evident, intramuscular injection of 50 μL of thePGE2-bupivacaine mixture is performed. Previous reports have shown thatthe proposed maximum dose of PGE2 20 μg (or 4 mg/kg) can be administeredto rodents without deleterious effects. Therefore a range of PGE2 dosesfrom 5, 10, and 20 μg are delivered to the GA. Delivery entails four12.5 μL injections, two into each GA muscle per leg (0.5 cc total). Ascontrols, GA muscles are injected with vehicle alone (i.e., PBS), 0.5%Bupivacaine alone, or PGE2 alone (5, 10, or 20 μg). In vivo MuSCactivity is assessed by bioluminescence imaging (BLI) every 2 days for 2weeks post-injury, and biweekly afterward for 3 months. The optimalPGE2-bupivacaine dose is the lowest effective dose with a saturatingeffect on muscle regeneration, as assessed by BLI. Endogenous MuSCpopulations are believed to be initially activated and then rapidlyproliferate, yielding an exponential increase in BLI signal that peaksand then plateaus when regeneration reaches homeostasis. 6 mice percondition (total 48 mice) are analyzed, to achieve 95% statisticalpower, based on a two-sided alpha of 0.05 and a standard deviation of6.24×10⁵ p/s/cm²/sr in BLI intensity (based upon prior data).

In summary, the experiments described in this section determine theformulation, or optimal dose of PGE2, that enhances expansion ofendogenous MuSC numbers in regeneration. The optimal dose is then usedin experiments described in the following section of this example.

Assessing long-term effects of a PGE2-bupivacaine formulation on muscleregeneration by immunohistology: The improvement of muscle regenerationmeasured by MuSC numbers and BLI is corroborated with histologicalanalysis of myofiber size, architecture and stem cell numbers in anindependent transgenic mouse line Pax7^(CreERT2) Rosa26-LSL-dtTOMATO.Using this line, MuSC activity is assessed, as is regeneration based onlineage tracing and assessment of the number of dtTOMATO+ myofiberspost-injury. The optimal dose of PGE2 established as described above isused in atrophic Pax7^(CreERT2); Rosa26-LSL-dtTOMATO mouse muscles.TOMATO signal is used to trace activated stem cells and theirdifferentiated progeny histologically in muscle fibers tissue sections.

Whereas the bioluminescence and TOMATO assays track expansion ofendogenous stem cells, they do not distinguish stem cells, subsets whichinclude progenitors, and differentiating myoblasts. To address thisneed, histological analysis and immunostaining is performed withantibodies to myogenic transcription factors that mark stem cells (e.g.,Pax7), progenitors (e.g., Myf5) and differentiated cells (e.g., MyoD) atthe end of each experiment. Also quantified is the contribution of MuSCsto myofibers and self-renewal to yield stem cells in niches in thesatellite cell position along the myofiber. Myofibers are identified byimmunostaining with antibodies to laminin and myosin heavy chain (MHC).Based on previous work, it is expected that soon after PGE2 treatmentthere will be a boost the proportion of stem cells relative to moredifferentiated cells in the GA muscle.

To analyze muscle reinnervation, the “reinnervation ratio” is scored asthe frequency of reinnervated neuromuscular junctions (NMJs). This ratioderives from the total NMJs labeled by neurofilament and synaptic vesselprotein (SV2) immunoreactivity (new NMJs) as a function of the frequencyof neuromuscular junctions labeled by α-bungarotoxin and Schwann cellGAP-43 immunoreactivity (total NMJs).

For a more quantitative assessment of the total GA muscle, FACS analysisof dissociated wildtype muscle tissue is performed to determine theratio of muscle stem cells (Pax7⁺) to activated (Pax7⁺/Myf5⁺) andcommitted (MyoD⁺/Myogenin⁺) myogenic cells. This is critical forestablishing that PGE2 is enhancing stem cell activity.

For these experiments, 6 mice per group are used: vehicle alone,bupivacaine alone, optimal PGE2 dose alone, and bupivacaine and optimalPGE2 dose together, for histological (n=24 mice) and for FACs analysis(n=24 mice), to achieve 95% statistical power. A non-parametric t-test(e.g., Mann-Whitney test) is used to assess the statistical significanceof all proposed animal experiments.

Example 6 Assessing Mouse Muscle Response to PGE2 by Assessing Strength,Architecture, and Muscle Volume

This example illustrates a study which can be used to evaluate musclefunction improvement in mice using multiple technologies. A novelhandheld microendoscope built by the Delp laboratory at Stanford (FIG.25) is employed that allows measurement of the contractile dynamics froma single motor unit in mice or humans. Force generation dynamics arecalculated based on the time course of sarcomere displacement. Inparallel, in vivo force measurements are made using an independenttechnique developed by the Blau and Delp labs at Stanford to evaluatestrength and assesses twitch and tetanic muscle force. Additionally,increases in muscle volume are measured by ultrasound to determine theextent of injured muscle recovery of muscle mass. These measurementsassess the efficacy of the PGE2-bupivacaine formulation in promotingmuscle regeneration in the atrophic GA mouse model. Histologicalassessment of the fiber sizes and extent of reinnervation is performedto cross-validate the outcomes based on endoscopy and ultrasoundstudies. Importantly, the microendoscopy and ultrasound assessmentmethodologies can be directly translated and applied to evaluate thehuman hand muscles of patients to assess baseline function andpost-intervention recovery.

Following the experiments described above in Example 5, as describedherein mice are assessed using ultrasound, microendoscopy, and forceassays to evaluate increases in muscle volume, architecture, andstrength. Using a novel handheld microendoscope, non-invasivemeasurements of the contractile dynamics of a single motor unit areperformed. Force generation is calculated based on the time course ofsarcomere displacement. In some instances, this assay is conductedweekly over an 8-week time-course. The aim is to assess the functionalimprovement of muscles that have received a combination of PGE2 andbupivacaine treatment compared to a control group. The volume of thetreated muscles is also evaluated by ultrasound analysis.

Evaluation of muscle function with measurements of strength usingmicroendoscopy: The ultimate test of the effects of a combination ofPGE2 and bupivacaine on muscle regeneration is to perform a functionalassay for muscle force increase. Using microendoscopes as small as 350micrometers, individual sarcomeres can be imaged in passive andactivated muscle, allowing for direct visualization of individualsarcomeres and length changes in a dynamic manner, a technologydeveloped in the Delp lab . As described herein, measurement is made ofthe contractile dynamics and force generating capacity of muscle tissueproduced by endogenous MuSCs after injection of the optimalPGE2-bupivacaine dose determined above in Example 5. Sarcomere length isalso assessed using minimally invasive optical microendoscopy in orderto observe second-harmonic frequencies of light generated in the musclefibers in mice after treatment. Striated skeletal muscles are comprisedof sarcomeres, the basic contractile units. Useful instruments forperforming these measurements include a laser-scanning microscope,adapted to allow the addition of a microendoscope for deep-tissueimaging, and an ultrashort-pulsed titanium-sapphire laser to generatesecond-harmonic signals. This minimally invasive technology can bereadily translated to the clinic for assessing parameters of musclefunction and can used to evaluate the efficacy of PGE2-bupivacainetreated abductor pollicis brevis (APB) in patients (see, Example 7below). Force is assessed 6 weeks post PGE2-bupivacaine treatment inmice treated as described above in Example 5. Accordingly, the optimalPGE2-bupivacaine dose not only yields the highest BLI signal but alsoyields the strongest force output compared to that of a PBS control. Asan alternative to the microendoscope, strength can be assessed in vivoby a force transducer technique established by the Blau laboratorypreviously. 6 independent mice per condition are analyzed to obtain 95%statistical power.

Evaluation of muscle volume by ultrasound imaging. Ultrasound imaging isa non-invasive method that can be used to assess muscle regenerationbased on muscle volume. Furthermore, ultrasound is a fast andnoninvasive method, allowing repeated measurements to be made over timeto evaluate the effects of compositions and methods of the presentinvention on muscle mass. Ultrasound imaging of the muscle is based onthe different acoustical impedance produced when the ultrasound beamencounters tissue. Atrophy and increased muscle mass due to regenerationcan be readily quantified by measuring muscle thickness, as thesonographic appearance of muscle is quite distinct from the surroundingfat, fibrous tissue, nerves, blood vessels and bone.

Neuromuscular atrophy due to CTS causes structural muscle changes thatcan be visualized with ultrasound. Atrophy can be objectified bymeasuring muscle thickness (i.e., the muscles become whiter on theultrasound image). Ultrasound is more sensitive in detectingfasciculations compared to electromyography (EMG) and clinicalobservations, because ultrasound can visualize a large muscle area anddeeper muscles, especially in the hand. With improving resolution andframe, smaller scale spontaneous muscle activity such as fibrillationscan be detected by ultrasound.

Example 7 Clinical Trial for the Treatment of Carpal Tunnel Syndrome

This example illustrates a clinical trial designed to determine thebenefit of compositions and methods of the present invention for thetreatment of patients with severe carpal tunnel syndrome (CTS). Theintervention for this trial tests the therapeutic strategy by injectinga PGE2-bupivacaine formulation into denervated abductor pollicis brevis(APB) muscles 2 months post carpal tunnel release. The followingoutcomes are assessed: 1) upper extremity function using the upper limbPROMIS assessment; 2) patient outcomes as assessed by a self-evaluationquestionnaire standardized by The Canadian Occupational Performance; 3)Moberg pickup test ; 4) ultrasound volume measurements; 5) functionalstrength using pinch strength using the “digital Pinch Dynamometer”device; and 6) determination of muscle mass by ultrasound and musclearchitecture by microendoscopy. Assessments are performed at 1, 3, and 6months post intervention. The studies described in this example canserve as a phase II clinical trial for the treatment of CTS muscleatrophy and provide a platform for the treatment of other nerve-relatedmuscle atrophies, which are a major problem for both combat casualtiesas well as the aging population.

Design: The study is a randomized placebo-controlled trial to assesswhether intramuscular PGE2 administration improves muscle recovery afterdenervation. This clinical trial that will yield two deliverables: (1)It will reveal if PGE2 improves denervated muscle regenerative capacity;(2) it will also test if PGE2 improves muscle function afterdenervation/re-innervation.

Study Population

Inclusion Criteria: Subjects who meet the following criteria areincluded in the trial:

-   -   Patient is scheduled for open carpal tunnel release at the Palo        Alto VA    -   Nerve conduction studies showing motor impact of the APB muscle        with distal motor latency to APB<6.5 ms    -   Persistent weakness of APB muscle 2 months after carpal tunnel        release as tested by tip pinch measurement

Exclusion criteria: subjects having one or more of the following areexcluded from participating in the trial:

-   -   Diagnosis of glaucoma    -   Inability to complete study forms (education, cognitive ability,        mental status, medical status).    -   Previous adverse reactions to prostaglandins.    -   Asthma    -   Systolic blood pressure greater than 170 mm/hg at time of        administration of intervention    -   Pregnancy    -   Unable to remain off NSAIDS for two days before and after        intervention    -   Persistent surgical site pain greater than 3 on 0-10 pain scale

Withdrawal: All patients approached for screening, the number agreeingto participate, the number providing informed consent, the numbercompleting the baseline evaluation measures, the number undergoingrandomization, and the number completing the trial in their assignedgroup are recorded. Patients choosing not to undergo randomization areasked if they will participate in the observational study identical tothe proposed clinical trial but without any active intervention. In thismanner an attempt is made to collect follow-up data from those notparticipating as well as those undergoing randomization to identify ifthe trial population differs significantly from those not participating.Patients may withdraw at any time.

Surgery: Surgery is an open carpal tunnel release. These procedures areperformed under local anesthetic with 1% lidocaine with epinephrine.This procedure uses a longitudinal incision on the palmar aspect of thehand. The patients are wrapped in light gauze without splintimmobilization and return 12 days for suture removal. At the 12-dayvisit the patients are taught scar massage and only follow up again ifthey are having difficulties.

Medications/Randomization: PGE2 (dinoprostone) that is in solution at a10 mg/ml dilution that comes in 0.5 ml ampule (Pfizer, UK) is used forthe intervention arm. Dinoprostone has been used for many years inpregnant women as a drug for induction of labor. This study repurposesthis clinically available medication. The most common side effects inhumans are largely pregnancy related: uterine rupture, and amnioticembolism. Other side effects reported include anaphylaxis, pyrexia,chest pain, arrhythmias, nausea as well as others. The half-life of thedrug is less than 5 minutes.

This medication is diluted with bupivacaine to the optimal dose asdirected by one or more preceding animal trials for optimal dosage.Matching placebo injections that are identical in appearance are mixedwithin a pharmacy. The randomization (i.e., assignment of subjects totreatment or control groups) is prepared using an R-program written bythe Stanford department of biostatistics. This program utilizes a“biased-coin” methodology that progressively alters the probability ofrandomization assignment to correct any imbalances in the groups ofpreviously randomized subjects. This “biased-coin” approach reduces thelikelihood of significant imbalances between treatment groups in allstrata while maintaining unpredictability in every treatment assignment.Randomization is done in a 1:1 ratio of treatment to placebo.

Recruitment: Subjects with APB weakness on physical exam are approachedfor recruitment and enrolled and pre-operative assessments completed.APB strength is assessed using tip pinch strength using dynamometry. Tippinch is a recommended measure for assessing APB strength in CTS. Tippinch is also the first pinch/grip test that shows improvement aftercarpal tunnel release. For inclusion in the trial, the APB strengthneeds to be 15% less than normative data or 3.84 kg in women and 5.78 kgin men.

Two months after surgery, subjects are re-evaluated. Those who havepersistent weakness then proceed with the intervention. This allows fortreatment of those who have failed to recover from nerve release alone.Persistent weakness is defined by using a force measure of the APB withthe patient having 15% less force than the contralateral side. Patientswith continued pain greater than 3 at the surgical site are thenexcluded. Pain greater than three is moderate pain and may interferewith force generation. Previous work suggests (minocycline trial) that6% will still have moderate pain at 3 months.

About 50% of the subjects have APB denervation. Of those 50% areexpected to have persistent weakness at 2 months .

Pre-operative Data Acquisition: After recruitment participants completea baseline assessment. This is accomplished prior to the carpal tunnelrelease. This assessment includes the measures listed below:

Demographic data: Ethnic origin, race, age, gender and medicalcomorbidities.

TABLE 3 Nerve conduction severity scoring Grade Definition 0 Noabnormality 1 CTS demonstrable only with most sensitive tests 2 Sensoryconduction slow but normal terminal motor latency 3 SNAP preserved withmotor slowing, distal motor latency to APB < 6.5 ms 4 SNAP absent butmotor response preserved, distal motor latency to APB < 6.5 ms 5Terminal latency to APB > 6.5 ms 6 Sensory and motor potentialseffectively unrecordable

This is a score that categorizes severity of the carpal tunnel usingnerve conduction studies. Nerve conduction studies are operatordependent and values can vary between sites. This assessment eliminatesthis problem by using values that are abnormal for that test for thattesting site instead of a precise value.

Upper Extremity PROMIS instrument: The PROMIS Upper extremity instrumentis a computer adaptive test, which draws items from the PROMIS item Bank(v1.0: PF) that has a collection of calibrated questions that define andquantify a particular symptom or functional problem. It uses an itemselection algorithm that enables the assessment program to choose arespondent's next item based on the response given to the respondent'scurrent item, thereby avoiding the presentation of redundant,irrelevant, or otherwise poorly targeted items. PROMIS scores arereported as T-scores, with a mean of 50 and a SD of 10; higher scoresrepresent higher levels of PF. This test has been evaluated and showedto have a reliability >0.95 for a representative US sample.

Canadian Occupational Performance Measure (COPM): This patient-centricexam measures the patient's perceived occupational performance in thearea of self-care, leisure and productivity. It is carried out in a5-step process nested within a semi-structured interview conducted by aprovider which typically takes 10-20 minutes to administer. The patientidentifies areas of difficulty and prioritizes them. The patient'sperformance and satisfaction are rated in the areas important to thepatient. The performance and satisfaction scores are measured for changeover time. The COPM has been shown to be responsive to change, with atwo-point improvement on performance scores recognized as clinicallysignificant.

Moberg pickup test: In this test, the thumb, index finger, and middlefinger are used to pick up 12 different objects, one at a time, atrandom. The patient puts them in an open box as quickly as possible. Thetime (in seconds) required to complete the task is measured and recordedas the end point. The is performed twice until all objects are pickedup, or until 30 seconds has been spent unsuccessfully attempting to pickup an identified object. The test is repeated with and without ablindfold. The same procedure will be done for the uninjured hand. Thiswill be scored as an index between the injured hand and the other hand.This test provides information on fine motor and sensory function of thehand.

Tip Pinch strength: Tip pinch strength is measured using a digital pinchdynamometer, used for measuring a patient's hand strength to evaluatethe degree of patient's APB muscle dysfunction. This tip pinch appliesforce of the thumb pulp to index pulp. The patient is seated with thetest arm at his/her side and the elbow flexed 90°. The palm faces downand pinch strength is measured between the pad of the thumb and the padand the index finger. The patient squeezes, holds, and releases. Thistest is performed in three consecutive measurements within 2 minutesinter-measurement interval.

Ultrasound volume measure of the APB: Ultrasound has been used to assessthe volume of the APB and the other small muscles in the hand.Ultrasound can detect several aspects of muscle. First, the ultrasoundcan identify the size of the muscle. This is routinely done by measuringthe cross sectional area (CSA). CSA of the APB is strongly correlatedwith muscle strength. The APB muscle is measured by placing the probe atthe proximal third of the first metacarpal bone. The images of the CSAare saved as bitmap image files and transferred to a personal computer.The analysis of the images is performed using Adobe Photoshop CS6Extended. The lasso tool is used to identify the muscle in question. TheCSA is computed using the analysis option. The fascia delineates themargins of APB muscles and allows separation of the APB from theopponens pollicis (an adjacent thenar muscle).

The muscle's echo intensity is also measured and is a sign ofdenervation. Indeed the echo intensity and homogeneity of the musclecorrelate with severity of CTS. The APB muscle is measured by ultrasoundusing B-mode with a 5-10 MHz transducer and the following equipmentsettings: 50 dB gain, 56 dB dynamic range, and 3 cm depth. The subjectsits with hands fully supinated. CSA is measured. All measures arerepeated three times and the mean is taken as the final measure.

The ten test: The ten test is an efficient method to assess sensationand is a marker of severity of the carpal tunnel syndrome. This test isperformed with the patient seated palm up. The patient is advised of the0-10 score of the test. The examiner uses the pulp of the indexfingertip and strokes lightly an area of normal sensation (often thecontralateral digit). The participant is instructed that this representsa score of 10 on the scale. Subsequently, the abnormal area and normalarea are stimulated simultaneously using identical pressure and theparticipant scores the stimulus on the affected limb (0-10) incomparison to the normal anchor area. From these measurements, a sensoryratio is derived.

Intervention: At 2 months after surgical release, patients arere-evaluated for persistent weakness of the APB on tip pinch strength.Those who continue to have weakness participate in the trial and arerandomized to placebo vs. combination of PGE2 and bupivacaine. The doseis extrapolated from the optimal dose determined in mice as describedabove in Examples 5 and 6. In preclinical preliminary studies, 10 μgPGE2 was administered per average GA muscle mass (135 mg). The dose inhumans can be extrapolated from the dosage used in mice since the drugis not being delivered systemically. The dose for use in the APB muscleis well below the dose clinically used as a drug to induce labor inpregnant women (i.e., 1-5 mg per treatment).

For the intervention, antiseptic technique is used and the skin isanesthetized with a weal of 1% lidocaine. Then 0.5 cc of either acombination of PGE2 and bupivacaine or a combination of saline andbupivacaine is injected in 4 aliquots into the APB muscle using a 30gauge needle. Patients are monitored for half an hour post-injection.The half-life of PGE2 is 5 minutes, thus 30 minutes is sufficient tomonitor for any acute reaction to the medication.

Exercise is an important component to muscle regeneration. All patientsare given an exercise program to strengthen their APB muscles withinstructions to perform exercises at least 10 minutes/day. Theraputty isprovided to each patient to assist with their home therapy program.Patients are contacted weekly to encourage them to perform theactivities every day.

Post-Surgery Data Acquisition: Patients are assessed at 4 months and 6months after surgery (2 and 4 months post-intervention). Post-operativeassessments will include:

-   1. Upper Extremity PROMIS-   2. COPM-   3. Moberg pickup test-   4. Ultrasound volume measure-   5. Tip Pinch strength-   6. Participation in formal physical therapy: each patient is asked    if they have worked with a physical therapist on their hand. Formal    physical therapy could potentially impact the results. Thus those    who receive physical therapy are excluded.-   7. Exercise compliance: each participant is asked how often they did    their exercises: daily, a few times per week, weekly or not at all.-   8. Adverse events: subjects are asked if they have had any health    issues since the last visit.

An exemplary subject timeline is shown in FIG. 26.

Microendoscopy: Two groups are selected to undergo microendoscopy. Thisis performed on 5 healthy volunteers at the beginning of the trial tounderstand healthy muscle architecture. Microendoscopy is also performedon 20 subjects pre-intervention and 4 months post-intervention to assessimprovement. Since this is a double-blind study, subjects will beincluded from both groups by random selection. Thus, it is expected thatat least 5 of each group (PGE2 vs. placebo) will be represented. Inprevious studies, this number has sufficed to yield significantfindings. The technique is the same for all. Subjects are seatedcomfortably with the hand at rest in supination. Antiseptic technique isused and ultrasound guidance is used to place the microendoscope intothe APB muscle. The ultrasound helps determine the muscle location andfiber orientation. The microendoscope is extracted slowly at 1 mmincrements. At least 3 different fibers are imaged.

Analysis

Endpoints and Measurements: The trial has three types of endpoints:feasibility endpoints, safety endpoints, and efficacy endpoints.

Primary Feasibility Endpoint: Feasibility endpoints are designed toprovide reliable measures of efficacy at completing study processes. Theprimary feasibility endpoint is the percentage of people screened thatcomplete the study in the group to which they were randomized.

Safety and Adverse Events Endpoint: Active capture of side effects isaccomplished during the administration of the medication and with afollow up phone call on post intervention day 2 specifically asking foredema, bruising or increased pain. Adverse events are recorded andreviewed bimonthly during the trial.

Efficacy Endpoints: The primary efficacy endpoint is force measure ofAPB using tip pinch strength in kg measured by dynamometry. Thesecondary endpoint includes changes in APB muscle cross sectional area.This is recorded using ultrasound and is measured in cm².

Analysis Populations: All randomized patients are included in anintention-to-treat analysis. Patients with 100% compliance are includedin a per-protocol analysis.

Background and Demographic Characteristics: Demographic and backgroundinformation is summarized with descriptive statistics (e.g., mean,standard deviation, percentages, and the like)

Analysis of Feasibility: A formal analysis of factors leading to failureto complete the study protocol is undertaken based on baseline variablesobtained at the time of consent. Patients are dichotomized according tothe primary feasibility endpoint (i.e., did they complete the study in agroup to which they were randomized). Logistic regression is used toidentify factors associated with failure to complete the study.

Analysis of Efficacy: Primary and secondary efficacy endpoints areanalyzed using linear regression models. Patient factors such as age,diabetes status, and NCS score will be controlled for. Study sample sizeand power are derived from this comparison (see below). Secondaryanalysis of the primary endpoint is corrected for multiple comparisons.Exploratory subgroup analysis is not corrected for multiple comparisons.

Analysis of Safety: Descriptive statistics and counts of the adverseevents associated with intervention are performed. Rates between placeboand intervention group are compared.

Methods for handling missing data and non-adherence to protocol: Primaryanalysis is intent-to-treat. Separate efficacy analysis on those withcomplete protocol adherence is also performed.

Evaluation of Conduct of trial (including accrual rates, data quality):The conduct of the trial is reviewed every 20 primary endpoint events.Feasibility endpoints are reviewed to ensure likelihood of trialcompletion. For these reviews accrual rates and cumulative statistics onprotocol violations will be prepared.

Subgroup Analyses: Subgroup analysis examines the treatment efficacy inpre-defined high risk subgroups defined by age greater than 70 andsevere entrapment score for nerve conduction studies.

Sample Size

Accrual estimates: Sixty subjects are enrolled over a 30-month period.This equates to approximately 2 patients per month.

Sample size justification: 60 patients are recruited for the study. Thebasis for the sample size is as follows. The Null hypothesis is nochange in force measurements of the APB between treatment groups. Thealternative hypothesis is that the combination of PGE2 and bupivacainesignificantly changes the force measurements of the APB.

A two-sample t-test with equal variance is used to test for differencesin force measurement of the APB between the placebo and PGE2-bupivacainegroups. A difference in total effect size is sought for primary outcomewith 80% power, based upon data from a previous study in mice with a10.0 N difference in tetanic force measurement. Therefore, the power iscalculated for testing the hypothesis using the Proc Power calculationusing the statistical analysis software (SAS version 9.4) with thefollowing parameters: Group Assignment=1:1, Pooled StandardDeviation=7.62 N, and a one-sided alpha of 0.05. The number of subjectsneeded per group to have 80% power with a one-sided alpha of 0.05 isdetermined to be 25 patients per group. If a 15% drop out is plannedfor, 29 patients are needed per group, or a total of 58 patients. Basedupon preclinical data, it is anticipated that the magnitude of theincrease in force in the treatment group will make a significantdifference to subjects when translated to the lateral pinch forcereported for CTS patients. Per published results, the anticipated forceincrease will allow subjects to perform certain daily basic tasks notpreviously possible, such as the ability to hold a fork or to pull up azipper (Smaby et al, 2004).

Additional Analyses: Several other outcomes can be assessed, includingchanges in COPM scores and time of Moberg pick up test. Both of theseare continuous variables and are compared using the Mann Whitney U test.Statistical analyses of APB function based on microendoscopymeasurements can also be performed.

Potential Pitfalls

Inadequate Recruitment: If difficulties are met in recruiting anadequate number of subjects, the recruitment pool can be expanded toinclude those with entrapment of the ulnar nerve at the elbow withintrinsic muscle wasting. This disease process is similar to carpaltunnel syndrome with wasting of multiple small muscles of the hand. Ifrecruitment is altered in this manner, changes to the protocol are made,as follows.

-   1) The timing of injection is 4 months after release to provide more    time for nerve regeneration.-   2) The muscle to undergo assessment is the first dorsal interossei.    The first dorsal interossei has also been measured with ultrasound,    with size being correlated with strength.-   3) The analysis plan still includes the assessments described above.    Of the ulnar nerve releases that are performed, about 50% have    atrophy.

Veteran population: In some instances, the study population may not berepresentative of the general population. For example, military veteransare older and have a higher male population. However, intervention withPGE2 appears to be particularly effective for the lack of regenerationin older mice. Thus, having an older population may be best suited forseeing the benefits of this intervention.

Military Benefit and Impact

PNI is a common combat injury that prevents many soldiers from returningto active duty. In addition, many older veterans have PNI fromcompression injuries that result in loss of ability to performactivities of daily living. Loss of muscle function after PNI continuesto be a difficult and unsolved question. Research to improve nerveregeneration continues but less has been done to improve muscleregeneration with re-innervation. A medication treatment to improve theamount of muscle recovery after nerve injury and repair is needed. Thepotential applications beyond this focused CTS trial are broad and wouldallow treatment of both the nerve and the muscle.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference.

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Example 8 Synergistic Effect of PGE2 Compound and Myotoxin Combinationin Muscle Regeneration

Pax7-CreERT2; Rosa-LSL-Luciferase mice (2-4 months old) were treatedwith tamoxifen for five consecutive days in order to obtain Pax7promoter expressing luciferase mice in vivo. One week later, baselinetetanic force of the tibialis anterior was measured using a foot plateforce measurement instrument before injection of drugs (timepoint day0). Mice were subsequently injected with 50 μl of vehicle (saline), themuscle stem cell activator prostaglandin E2 (PGE2, 20 μg), the musclestem cell expansion agent bupivacaine (BPV, 0.25%) or the combinationdrug (bupivacaine 0.25% together with PGE2 20 μg) into the Tibialisanterior (TA) muscle. FIG. 27A shows bioluminescence (BLI, measured asradiance) measured every 3 days for 2 weeks to measure muscle stem cellexpansion. FIG. 27B shows the resulting tetanic force measured at week 4from the same mice, where the percent difference to baseline force wascalculated. FIG. 27C: at 4 weeks (endpoint) the TA was isolated, and thespecific force (mN/mm²) was obtained based on the physiologicalcross-sectional area (PCSA) calculated by the muscle length, weight andpennation angle. The specific force and the percent difference oftetanic force were significantly increased for the combination drugcompared to the vehicle and both of the small molecules injected alone.*P<0.05, **P<0.001. ANOVA test for group comparisons and significantdifference for endpoint by Fisher's test (FIG. 27A). ANOVA test withBonferroni correction for multiple comparisons (FIG. 27B, FIG. 27C).Data are shown as means±SEM.

The results reveal that a combination of a PGE2 compound with a myotoxinsuch as bupivacaine has a synergistic effect on muscle regeneration. Asshown in FIG. 27A, the combination of PGE2 and bupivacaine inducesmuscle stem cell expansion that is greater than that of PGE2 alone orbupivacaine alone. Importantly, the observed muscle stem cell expansionis greater than the sum of muscle stem cell expansion for PGE2 alone,and bupivacaine alone. The synergistic effect on muscle regeneration isalso confirmed with another assay. As shown in FIG. 27B, the increase intetanic force for muscles treated with PGE2 and bupivacaine combined isgreater than the sum of the increase in tetanic force for musclestreated with PGE2.

What is claimed is:
 1. A method of treating a muscle condition, themethod comprising: (a) administering a prostaglandin E2 (PGE2) compoundto a subject in need thereof and (b) administering a myotoxin to thesubject, wherein the administering is by intramuscular administration.2. The method of claim 1, wherein the PGE2 compound is selected from thegroup consisting of PGE2, a PGE2 derivative, a PGE2 prodrug, a PGE2receptor agonist, a compound that attenuates PGE2 catabolism, a compoundthat neutralizes PGE2 inhibition, a derivative thereof, an analogthereof, and a combination thereof.
 3. The method of claim 2, whereinthe PGE2 compound is PGE2.
 4. The method of claim 2, wherein the PGE2compound is a PGE2 derivative, and the PGE2 derivative comprises16,16-dimethyl prostaglandin E2.
 5. The method of claim 2, wherein thePGE2 compound is a compound that attenuates PGE2 catabolism, and thecompound that attenuates PGE2 catabolism comprises a compound,neutralizing peptide, or neutralizing antibody that inactivates orblocks 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivates orblocks a prostaglandin transporter (PGT or SLCO2A1).
 6. The method ofclaim 1, wherein the myotoxin is selected from the group consisting ofan anesthetic, a divalent cation, snake venom, lizard venom, bee venom,and a combination thereof.
 7. The method of claim 6, wherein themyotoxin is an anesthetic, and the anesthetic is selected from the groupconsisting of an amino-amide anesthetic, an amino-ester anesthetic, anda combination thereof.
 8. The method of claim 6, wherein the anestheticis an amino-amide anesthetic, and the amino-amide anesthetic is selectedfrom the group consisting of bupivacaine, levobupivacaine, articaine,ropivacaine, butanilicaine, carticaine, dibucaine, etidocaine,lidocaine, mepivacaine, prilocaine, trimecaine, and a combinationthereof.
 9. The method of claim 6, wherein the anesthetic is anamino-ester anesthetic, and the amino-ester anesthetic is selected fromthe group consisting of an aminobenzoic acid ester anesthetic, a benzoicacid ester anesthetic, and a combination thereof.
 10. The method ofclaim 9, wherein the amino-ester anesthetic is an aminobenzoic acidester anesthetic, and the aminobenzoic acid ester anesthetic is selectedfrom the group consisting of benzocaine, butacaine, butamben,chloroprocaine, dimethocaine, lucaine, meprylcaine, metabutethamine,metabutoxycaine, nitracaine, orthocaine, propoxycaine, procaine,proxymetacaine, risocaine, tetracaine, and a combination thereof. 11.The method of claim 9, wherein the amino-ester anesthetic is a benzoicacid ester anesthetic, and the benzoic acid ester anesthetic is selectedfrom the group consisting of amylocaine, cocaine, cyclomethycaine,α-eucaine, β-eucaine, hexylcaine, isobucaine, piperocaine, and acombination thereof.
 12. The method of claim 6, wherein the myotoxin isa snake venom or a lizard venom, and the snake venom or the lizard venomis selected from the group consisting of notexin, cardiotoxin,bungarotoxin, and a combination thereof.
 13. The method of claim 6,wherein the myotoxin is a divalent cation, and the divalent cation isselected from the group consisting of Ba²⁺, Sr²⁺, Mg²⁺, Ca²⁺, Mn²⁺,Ni²⁺, Co²⁺, a salt thereof, and a combination thereof.
 14. The method ofclaim 1, wherein the PGE2 compound is PGE2 and/or 16,16-dimethylprostaglandin E2, and the myotoxin is bupivacaine.
 15. The method ofclaim 1, wherein the muscle condition comprises muscle damage, injury,or atrophy.
 16. The method of claim 1, wherein the intramuscularadministration comprises an intramuscular injection into a skeletalmuscle.
 17. The method of claim 1, wherein intramuscular administrationcomprises an acute exposure, an intermittent exposure, a chronicexposure, or a continuous exposure, to the subject.
 18. The method ofclaim 1, wherein the intramuscular administration comprises anintramuscular injection into a muscle that is injured, damaged oratrophied.
 19. The method of claim 1, wherein the method comprisesadministering the prostaglandin E2 (PGE2) compound and administering themyotoxin together in a single formulation to the subject.
 20. The methodof claim 1, wherein the method comprises administering the prostaglandinE2 (PGE2) compound and administering the myotoxin sequentially inseparate formulations to the subject.
 21. The method of claim 20,wherein the method comprises administering the prostaglandin E2 (PGE2)compound before administering the myotoxin to the subject.
 22. Themethod of claim 20, wherein the method comprises administering theprostaglandin E2 (PGE2) compound after administering the myotoxin to thesubject.
 23. The method of claim 1, the method comprising administeringthe prostaglandin E2 (PGE2) compound with a pharmaceutically suitableexcipient and administering the myotoxin with a pharmaceuticallysuitable excipient to the subject in need thereof by intramuscularadministration.
 24. The method of claim 1, the method comprisingadministering the prostaglandin E2 (PGE2) compound and the myotoxin withthe pharmaceutically suitable excipient in a single formulation to thesubject in need thereof by intramuscular administration.
 25. The methodof claim 1, the method comprising administering the prostaglandin E2(PGE2) compound with a pharmaceutically suitable excipient andadministering the myotoxin with a pharmaceutically suitable excipientsequentially in separate formulations to the subject in need thereof byintramuscular administration.
 26. The method of claim 1, wherein theintramuscular administration comprises an intramuscular injection intothe muscle with the muscle condition.